Treatment of upper face lines

Abstract

The present invention provides a modified BoNT / A for use in the treatment of facial wrinkles wherein the modified BoNT / A is administered by intramuscular injection at a plurality of sites on the subject's face wherein the modified BoNT / A is administered at a unit dose per site of greater than 1754 pg (e.g. Greater than 1800 pg) of the modified BoNT / A, wherein the plurality of positions are selected from: a maximum of two positions of the rugobrow muscle and a maximum of one position of the norbrobrow muscle, a maximum of five positions for treatment of the interbrow lines, a maximum of five positions of the frontal muscle, a maximum of three positions for treatment of the forehead lines, and an orbicular orbicular muscle, a maximum of three positions for treatment of the outer canthi, and a maximum of three positions for treatment of the orbicular orbicular muscle. Wherein the total dose of the modified BoNT / A administered during treatment is up to 88,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain (HN domain), and a BoNT / B receptor binding domain (HC domain).

Description

Field of the Invention

[0001] The present invention relates to cosmetic treatments (eg, for treating hyperfunctional facial lines). More particularly, the present invention provides methods of cosmetic treatment comprising administering a long-acting neurotoxin, and more particularly to methods of treating cosmetic imperfections using a long-acting botulinum toxin. Background of the Invention

[0003] Hyperfunctional facial lines are common aesthetic defects and include, but are not limited to: glabellar lines, lateral canthal lines, forehead lines and wrinkles, crow's feet, glabellar lines, nasolabial folds, lip lines, and marionette lines. Upper facial lines can occur in the forehead, glabellar, and lateral orbital regions. Frowning expressions may also cause wrinkles to appear in the glabellar and forehead regions, while smiling expressions may also cause wrinkles to appear in the lateral canthal region. Hyperprominent lines in this area are often misinterpreted as fatigue, causing patients to become very distressed about their appearance. In reality, hyperprominent facial lines occur due to the functional pull of the underlying muscles, which ultimately causes the skin to wrinkle. In the glabellar complex, these muscles include the corrugator, procerus, and depressor supercilii, while the orbicularis oculi is responsible for producing lateral canthal lines.

[0004] Over the past few decades, as the aging population has continued to grow, there has been an increased demand for cosmetic procedures to reverse the appearance of aging, especially on the face. The increased concern about appearance has led to the development of many different products and procedures, such as surgery, various types of resurfacing, and the use of fillers. Since none of these methods are completely risk-free, continued research is needed to provide the safest and most effective treatments for facial aging.

[0005] While there are five factors that interact to cause facial aging, there are primarily two factors (skin and underlying muscles) that interact to cause more noticeable lines and folds rather than wrinkles. Many treatments have been developed to treat wrinkles and treat the skin factors of lines and folds, including various types of resurfacing, dermatologic products, and injectables for soft tissue augmentation.

[0006] Another cosmetic approach to treating facial lines is to administer neurotoxins, specifically Clostridium botulinum neurotoxin, to the underlying muscles of the facial skin. It is a pharmaceutical product containing the drug BoNT / A hemagglutinin complex (BTX-A-HAC) isolated and purified from Clostridium botulinum type A strain. There are several other pharmaceutical BoNT / A products on the market that are naturally produced by Clostridium botulinum.

[0007] BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve terminals, thereby blocking cholinergic transmission at the neuromuscular junction, resulting in decreased muscle contraction and decreased muscle tone, resulting in relaxation of the injected muscle.

[0008] Approved for the treatment of glabellar lines, up to a total dose of 50 units, injected into the corrugator and procerus muscles (see Figure 1 ). The clinical effects of Dysport can last up to four months. Clinical studies have shown that repeated dosing up to four times can maintain efficacy. However, Dysport should not be applied more frequently than once every three months. To date, Dysport is not FDA-approved for the treatment of forehead lines and lateral canthal lines.

[0009] In order to avoid systemic nervous system effects, many Clostridial toxin-based cosmetic treatments utilize Clostridial toxin therapeutics administered directly to a given target site (e.g., target tissue). One concern when administering Clostridial toxin-based therapeutics in this manner is that the toxin can diffuse from the site of administration into surrounding tissues or into the systemic circulation. Diffusion of toxins from target tissues is believed to be a cause of adverse side effects, which in extreme cases can be life-threatening. This can be a particularly concerning issue when Clostridial toxin therapeutics (e.g., BoNT therapeutics) are used at high doses, high concentrations, and high injection volumes. Adverse reactions reported for commercial BoNT / A therapeutics associated with this issue include fatigue, generalized muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and dyspnea. Swallowing and breathing difficulties can be life-threatening, and there have been reports of toxin diffusion leading to death.

[0010] The present invention overcomes one or more of the problems set forth above. Summary of the invention

[0011] The present inventors have unexpectedly discovered that modified BoNT / A has particular utility in the cosmetic treatment of facial wrinkles. The modified BoNT / A may include a BoNT / A light chain and a translocation domain and a BoNT / B receptor binding domain (H C domain), which results in the modified BoNT / A exhibiting increased retention at the site of administration (reduced diffusion from the site of administration) and increased duration of action (e.g., 6-9 months). Advantageously, the modified BoNT / A has similar properties to unmodified BoNT / A (e.g., ) compared to the modified BoNT / A described herein. This improved safety can be represented by the high safety ratio of the modified BoNT / A described herein. The modified BoNT / A can include modification of one or more surface exposed amino acid residues, resulting in an increased net positive charge. The increased charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and the cell surface. This, in turn, increases retention of the drug at the site of administration (reduces diffusion from the site of administration), thereby extending the duration of action (e.g., 6-9 months).

[0012] Based on the preclinical data herein (see Example 11), it has been shown that a higher total amount of modified BoNT / A can be administered to a subject while achieving comparable efficacy to unmodified BoNT / A (e.g., ) with a similar safety profile. Thus, modified BoNT / A can be injected into more muscles / sites in the cosmetic treatment of facial wrinkles before reaching a maximum total dose. This is an important and advantageous finding and results in improved cosmetic treatments for facial wrinkles while providing clinicians with a wider range of treatment options. Treatment can be improved because compared to using unmodified BoNT / A (e.g. ) treatment, it provides longer-lasting treatment (resulting in less frequent dosing) and / or can be customized to the subject, for example, enabling the clinician to apply at a specific site based on the subject's aesthetic requirements. The treatment of the present invention is improved over conventional treatment regimens.

[0013] In addition, the present invention provides a convenient, safe and effective single dose unit and a total (maximum) dose that can be safely applied in a single treatment. The present invention also provides corresponding guidance, which guides the number of times the dosage unit can be applied to the muscle (including the number of injection sites per muscle) without causing toxicity to the patient. Therefore, treatment according to the present invention is much simpler for clinicians and helps to avoid underdosage and / or overdosage. Summary of the invention

[0015] In one aspect, the invention provides a modified BoNT / A for use in treating facial wrinkles (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0016] wherein the modified BoNT / A is administered at a unit dose of greater than 73 units of the modified BoNT / A per site, and wherein 1 unit is the calculated median lethal dose (LD50) corresponding to mice 50 ),

[0017] Wherein, the plurality of sites are selected from:

[0018] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0019] Up to five sites on the frontalis muscle to treat forehead lines; and

[0020] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outside of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0021] wherein the total dose of modified BoNT / A administered during the treatment period is up to 3,661 units, and

[0022] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0023] Also provided are corresponding uses (in the manufacture of a medicament) and methods for treating facial lines.

[0024] In a related aspect, the invention provides a method of treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of sites on the face of a subject by intramuscular injection,

[0025] wherein the modified BoNT / A is administered at a unit dose of greater than 73 units of the modified BoNT / A per site, and wherein 1 unit is the calculated median lethal dose (LD50) corresponding to mice 50 ),

[0026] Wherein, the plurality of sites are selected from:

[0027] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0028] Up to five sites on the frontalis muscle to treat forehead lines; and

[0029] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0030] wherein the total dose of modified BoNT / A administered during the treatment period is up to 3,661 units, and

[0031] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0032] In another related aspect, the present invention provides a use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0033] wherein the modified BoNT / A is administered at a unit dose of greater than 73 units of the modified BoNT / A per site, and wherein 1 unit is the calculated median lethal dose (LD50) corresponding to mice 50 ),

[0034] Wherein, the plurality of sites are selected from:

[0035] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0036] Up to five sites on the frontalis muscle to treat forehead lines; and

[0037] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0038] wherein the total dose of modified BoNT / A administered during the treatment period is up to 3,661 units, and

[0039] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0040] In one aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0041] wherein the modified BoNT / A is administered at a unit dose of greater than 73 units of the modified BoNT / A per site, and wherein 1 unit is the calculated median lethal dose (LD50) corresponding to mice 50 ),

[0042] Wherein, the plurality of sites are selected from:

[0043] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0044] Up to five sites on the frontalis muscle to treat forehead lines; and

[0045] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0046] Preferably, the plurality of sites comprises (or consists of) at most two sites of the corrugator supercilii muscle and one site of the procerus supercilii muscle for treating glabellar lines,

[0047] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 1144 units (e.g., up to 1019 units), and

[0048] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0049] In a related aspect, the invention provides a method of treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of locations on the face of a subject by intramuscular injection,

[0050] wherein the modified BoNT / A is administered at a unit dose of greater than 73 units of the modified BoNT / A per site, and wherein 1 unit is the calculated median lethal dose (LD50) corresponding to mice 50 ),

[0051] Wherein, the plurality of sites are selected from:

[0052] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0053] Up to five sites on the frontalis muscle to treat forehead lines; and

[0054] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0055] Preferably, wherein the plurality of sites comprises (or consists of) at most two sites of the corrugator supercilii muscle and at most one site of the procerus supercilii muscle for treating glabellar lines,

[0056] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 1144 units (e.g., up to 1019 units), and

[0057] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0058] In another related aspect, the present invention provides a use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0059] wherein the modified BoNT / A is administered at a unit dose of greater than 73 units of the modified BoNT / A per site, and wherein 1 unit is the calculated median lethal dose (LD50) corresponding to mice 50 ),

[0060] Wherein, the plurality of sites are selected from:

[0061] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0062] Up to five sites on the frontalis muscle to treat forehead lines; and

[0063] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0064] Preferably, wherein the plurality of sites comprises (or consists of) at most two sites of the corrugator supercilii muscle and at most one site of the procerus supercilii muscle for treating glabellar lines,

[0065] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 1144 units (e.g., up to 1019 units), and

[0066] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0067] In another aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0068] wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site,

[0069] Wherein, the plurality of sites are selected from:

[0070] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0071] Up to five sites on the frontalis muscle to treat forehead lines; and

[0072] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0073] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 88,000 (e.g., up to 80,000 pg), and

[0074] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0075] In a related aspect, the invention provides a method of treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of sites on the face of a subject by intramuscular injection,

[0076] wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site,

[0077] Wherein, the plurality of sites are selected from:

[0078] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0079] Up to five sites on the frontalis muscle to treat forehead lines; and

[0080] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0081] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 88,000 (e.g., up to 80,000 pg), and

[0082] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0083] In another related aspect, the present invention provides a use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0084] wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (preferably greater than 1800 pg, more preferably greater than 2555 pg) of modified BoNT / A per site,

[0085] Wherein, the plurality of sites are selected from:

[0086] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0087] Up to five sites on the frontalis muscle to treat forehead lines; and

[0088] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0089] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 88,000 (e.g., up to 80,000 pg), and

[0090] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0091] In another aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0092] wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site,

[0093] Wherein, the plurality of sites are selected from:

[0094] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0095] Up to five sites on the frontalis muscle to treat forehead lines; and

[0096] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0097] Preferably, wherein the plurality of sites comprises (or consists of) at most two sites of the corrugator supercilii muscle and at most one site of the procerus supercilii muscle for treating glabellar lines,

[0098] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 27,500 (e.g., up to 25,000 pg), and

[0099] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0100] In a related aspect, the invention provides a method of treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of sites on the face of a subject by intramuscular injection,

[0101] wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site,

[0102] Wherein, the plurality of sites are selected from:

[0103] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0104] Up to five sites on the frontalis muscle to treat forehead lines; and

[0105] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0106] Preferably, wherein the plurality of sites comprises (or consists of) at most two sites of the corrugator supercilii muscle and at most one site of the procerus supercilii muscle for treating glabellar lines,

[0107] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 27,500 (e.g., up to 25,000 pg), and

[0108] The modified BoNT / A includes a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (HC domain).

[0109] In another related aspect, the present invention provides a use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection to multiple sites on the face of a subject,

[0110] wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (preferably greater than 1800 pg, more preferably greater than 2555 pg) of modified BoNT / A per site,

[0111] Wherein, the plurality of sites are selected from:

[0112] Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0113] Up to five sites on the frontalis muscle to treat forehead lines; and

[0114] Up to three sites on the orbicularis oculi muscle to treat lateral canthal wrinkles (e.g., up to three sites on the outer part of the orbicularis oculi muscle to treat lateral canthal wrinkles),

[0115] Preferably, the plurality of sites comprises (or consists of) at most two sites of the corrugator supercilii muscle and at most one site of the procerus supercilii muscle for treating glabellar lines,

[0116] wherein the total dose of the modified BoNT / A administered during the treatment period is up to 27,500 pg (e.g., up to 25,000 pg), and

[0117] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0118] The facial lines are most preferably referred to as "upper facial lines", and (as reflected in the above aspects) include glabellar lines, forehead lines, and lateral canthal lines. The present invention may be directed to treating any one of the facial lines (e.g., upper facial lines) or any combination thereof. For example, all three of glabellar lines, forehead lines, and lateral canthal lines may be treated by the methods of the present invention. Nevertheless, specific types of facial lines may be targeted. For example, the facial lines treated may be glabellar lines. Additionally, or alternatively, the facial lines treated may be forehead lines. Additionally, or alternatively, the facial lines treated may be lateral canthal lines.

[0119] The present invention can treat specific facial line combinations. Examples of facial line combinations include:

[0120] frown lines and forehead lines;

[0121] Glabellar lines and lateral canthal lines;

[0122] Forehead lines and lateral canthal lines.

[0123] A preferred combination of facial lines treated by the present invention may be glabellar lines and lateral canthal lines.

[0124] Reference to "muscle" refers to the affected muscle which contributes (eg, through excessive tightness, tension, or tone) to the underlying aesthetic condition targeted by the present invention.

[0125] The first group of muscles is defined by the procerus muscles, the second group of muscles is defined by the corrugator supercilii muscles, the third group of muscles is defined by the orbicularis oculi muscles, and the fourth group of muscles is defined by the frontalis muscles.

[0126] Throughout this disclosure, the term "up to two sites in the corrugator supercilii muscle" can be used interchangeably with the term "up to two sites in each corrugator supercilii muscle," noting that there are two corrugator supercilii muscles. Thus, a modified BoNT / A can be administered at up to four sites in the corrugator supercilii muscle, for example, provided that the modified BoNT / A is administered at up to two sites in each corrugator supercilii muscle.

[0127] The corrugator muscle is also called the corrugator supercilli.

[0128] The site of the orbicularis oculi muscle (to which a unit dose of modified BoNT / A is administered) is preferably located at the outer part of the orbicularis oculi muscle. The term "outer part of the orbicularis oculi muscle" is used interchangeably with the term "outer superior orbicularis oculi muscle."

[0129] Administration at "up to three sites in the orbicularis oculi muscle" may include:

[0130] a) administering a unit dose of a modified BoNT / A to the lateral superior orbicularis oculi muscle (e.g., near the subject's first eye);

[0131] b) administering a unit dose of the modified BoNT / A to the medial superior orbicularis oculi muscle (e.g., near the subject's first eye); and

[0132] c) administering a unit dose of the modified BoNT / A to the lateral inferior orbicularis oculi muscle (eg, near the subject's first eye).

[0133] Administration at "up to three sites in the orbicularis oculi muscle" may include:

[0134] a) administering a unit dose of a modified BoNT / A to the lateral superior orbicularis oculi muscle (e.g., near the subject's first eye);

[0135] b) administering a unit dose of a modified BoNT / A to a first site of the lateral inferior orbicularis oculi muscle (e.g., near the subject's first eye); and

[0136] c) administering a unit dose of the modified BoNT / A to a second (different) site on the lateral inferior orbicularis oculi muscle (eg, near the subject's first eye).

[0137] Throughout this disclosure, the term "up to three sites in the orbicularis oculi muscle" may be used interchangeably with the term "up to three sites in each of the orbicularis oculi muscles," noting that there are two orbicularis oculi muscles. Similarly, throughout this disclosure, the term "up to three sites outside of the orbicularis oculi muscle" may be used interchangeably with the term "up to three sites in each of the orbicularis oculi muscles, located outside of the orbicularis oculi muscle," noting that there are two orbicularis oculi muscles. Thus, a modified BoNT / A can be administered to up to six sites in the orbicularis oculi muscle, for example, provided that the modified BoNT / A is administered to up to three sites in each of the orbicularis oculi muscles.

[0138] Administration at "up to three sites in the orbicularis oculi muscle" may include:

[0139] a) administering a unit dose of modified BoNT / A to each lateral superior orbicularis oculi muscle;

[0140] b) administering a unit dose of a modified BoNT / A to each medial superior orbicularis oculi muscle; and

[0141] c) Administering a unit dose of modified BoNT / A to each lateral inferior orbicularis oculi muscle.

[0142] Administration at "up to three sites in the orbicularis oculi muscle" may include:

[0143] a) administering a unit dose of a modified BoNT / A to each outer superior orbicularis oculi muscle (e.g., near the subject's first eye);

[0144] b) administering a unit dose of a modified BoNT / A to a first site of the lateral inferior orbicularis oculi muscle (e.g., near the subject's first eye); and

[0145] c) administering a unit dose of the modified BoNT / A to a second (different) site on each lateral inferior orbicularis oculi muscle (eg, near the subject's first eye).

[0146] When referring to the "upper" orbicularis oculi muscle, it is the orbicularis oculi muscle of the upper eyelid. Likewise, when referring to the "lower" orbicularis oculi muscle, it is the orbicularis oculi muscle of the lower eyelid.

[0147] The term "medial" (e.g., in the context of anatomy) is understood by those skilled in the art to mean toward the midline of the body. Similarly, the term "lateral" (e.g., in the context of anatomy) is understood by those skilled in the art to mean away from the midline of the body. Thus:

[0148] - The "lateral" superior orbicularis oculi muscle refers to the site of the orbicularis oculi muscle on the upper eyelid, away from the midline of the body

[0149] - The "medial" superior orbicularis oculi muscle refers to the site of the orbicularis oculi muscle of the upper eyelid, located in the midline of the body

[0150] nearby

[0151] -The "lateral" inferior orbicularis oculi muscle refers to the site of the orbicularis oculi muscle in the lower eyelid, away from the midline of the body.

[0152] The term "lateral superior orbicularis oculi muscle" may be used synonymously with the term "external orbicularis oculi muscle of the upper eyelid." The term "medial superior orbicularis oculi muscle" may be used synonymously with the term "internal orbicularis oculi muscle of the upper eyelid." The term "lateral inferior orbicularis oculi muscle" may be used synonymously with the term "external orbicularis oculi muscle of the lower eyelid."

[0153] The orbicularis oculi muscle is composed of the "pretarsal part" and the "preorbital septum part" (both are injectable). The orbicularis oculi muscle is composed of the "orbital part".

[0154] Preferably, the unit dose administered to the orbicularis oculi muscle may be administered to the orbital portion of the muscle, more preferably to the orbital portion external (e.g., lateral) to the orbicularis oculi muscle. Thus, the plurality of sites for treating lateral canthal lines may include (or consist of) up to three sites in the orbital portion external (e.g., lateral) to the orbicularis oculi muscle.

[0155] Administration to the lateral superior orbicularis oculi muscle may mean administration to the lateral "pretarsal" superior orbicularis oculi muscle, or administration to the lateral "preseptal" superior orbicularis oculi muscle.

[0156] Administration to the medial superior orbicularis oculi muscle may mean administration to the medial superior "pretarsal" orbicularis oculi muscle, or administration to the medial superior "preseptal" orbicularis oculi muscle.

[0157] Administration to the lateral inferior orbicularis oculi muscle may mean administration to the lateral inferior "pretarsal" orbicularis oculi muscle, or administration to the lateral inferior "preseptal" orbicularis oculi muscle.

[0158] When administered to the lateral superior orbicularis oculi muscle, the unit dose may be administered to the anterior portion of the orbital septum (in other words, to the lateral "pre-septal" superior orbicularis oculi muscle). When administered to the medial superior orbicularis oculi muscle, the unit dose may be administered to the anterior portion of the orbital septum (in other words, to the medial superior "pre-septal" orbicularis oculi muscle). When administered to the lateral inferior orbicularis oculi muscle, the unit dose may be administered to the anterior portion of the orbital septum (in other words, to the lateral inferior "pre-septal" orbicularis oculi muscle).

[0159] The number of sites to which the modified BoNT / A is applied may vary depending on the facial lines to be treated.

[0160] For example, the facial lines to be treated can be glabellar lines, and the term "multiple sites" can mean up to three sites, wherein the multiple sites include (or consist of) up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for treating the glabellar lines. Preferably, two unit doses of the modified BoNT / A are administered to two different sites on the corrugator supercilii muscle, and one unit dose is administered to one site on the procerus supercilii muscle for treating the glabellar lines (e.g., such that three unit doses are administered to three different sites). Glabellar lines on one side of the face can be treated, and the term "multiple sites" can mean up to three sites as defined above, such that the subject receives up to three unit doses of the modified BoNT / A.

[0161] The facial lines to be treated can be glabellar lines, and the term "multiple sites" can mean up to six sites, wherein a unit dose of the modified BoNT / A acts on up to two sites on each corrugator supercilii muscle and up to one site on the procerus supercilii muscle for treating glabellar lines. Preferably, two unit doses of the modified BoNT / A are administered to two different sites on each corrugator supercilii muscle and one unit dose is administered to one site on the procerus supercilii muscle for treating glabellar lines (e.g., such that five unit doses are administered to five different sites). Glabellar lines on both sides of the face can be treated, and the term "multiple sites" can mean up to six sites as defined above, such that the subject receives up to six unit doses of the modified BoNT / A.

[0162] The facial lines to be treated may be forehead lines, and the term "multiple sites" may refer to a maximum of two, three, four or five sites of the frontalis muscle for treating forehead lines. Preferably, five unit doses are administered to five different sites of the frontalis muscle for treating forehead lines (e.g., such that five unit doses are administered to five different sites).

[0163] The facial lines to be treated can be lateral canthal lines, and the term "multiple sites" can mean up to two or three sites of the orbicularis oculi muscle (e.g., up to two or three sites outside the orbicularis oculi muscle). Preferably, three unit doses of the modified BoNT / A are administered to three different sites of the orbicularis oculi muscle for treating lateral canthal lines (e.g., such that three unit doses are administered to three different sites). Lateral canthal lines on one side of the face can be treated, and the term "multiple sites" can mean up to three sites as defined above, such that the subject receives up to three unit doses of the modified BoNT / A.

[0164] The facial lines to be treated can be lateral canthal lines, and the term "multiple sites" can mean up to two or three sites per orbicularis oculi muscle (e.g., up to two or three sites per orbicularis oculi muscle that are located outside the orbicularis oculi muscle). Preferably, three unit doses of the modified BoNT / A are administered to three different sites per orbicularis oculi muscle to treat the lateral canthal lines (e.g., such that six unit doses are administered to six different sites). The lateral canthal lines on both sides of the face can be treated, and the term "multiple sites" can mean up to six sites as defined above, such that the subject receives up to six unit doses of the modified BoNT / A.

[0165] The facial lines to be treated can be glabellar lines and forehead lines, and the term "plurality of sites" can mean up to eight sites, wherein the plurality of sites includes (or consists of) up to two sites of the corrugator supercilii muscle and up to one site of the procerus procerus muscle for treating glabellar lines, and up to five sites of the frontalis muscle for treating forehead lines. Preferably, two unit doses of the modified BoNT / A are administered to two different sites of the corrugator supercilii muscle, and one unit dose is administered to one site of the procerus procerus muscle for treating glabellar lines, and five unit doses are administered to five different sites of the frontalis muscle for treating forehead lines (e.g., such that eight unit doses are administered to eight different sites).

[0166] The facial lines to be treated can be glabellar lines and forehead lines, and the term "plurality of sites" can mean up to ten sites, wherein the plurality of sites includes (or consists of) up to two sites on each of the corrugator supercilii muscle and up to one site on the procerus procerus muscle for treating glabellar lines, and up to five sites on the frontalis muscle for treating forehead lines. Preferably, two unit doses of the modified BoNT / A are administered to two different sites on each of the corrugator supercilii muscle, and one unit dose is administered to one site on the procerus procerus muscle for treating glabellar lines, and five unit doses are administered to five different sites on the frontalis muscle for treating forehead lines (e.g., such that ten unit doses are administered to ten different sites).

[0167] The facial lines to be treated can be glabellar lines and lateral canthal lines, and the term "plurality of sites" can mean up to three, four, five, or six sites, wherein the plurality of sites includes (or consists of) up to two sites of the corrugator supercilii muscle and up to one site of the procerus procerus muscle for treating glabellar lines, and up to two or three sites of the orbicularis oculi muscle (e.g., up to two or three sites outside the orbicularis oculi muscle) for treating lateral canthal lines. Preferably, two unit doses of the modified BoNT / A are administered to two different sites of the corrugator supercilii muscle, and one unit dose is administered to one site of the procerus procerus muscle for treating glabellar lines, and three unit doses of the modified BoNT / A are administered to three different sites of the orbicularis oculi muscle for treating lateral canthal lines (e.g., six unit doses are administered to six different sites).

[0168] The facial lines to be treated can be glabellar lines and lateral canthal lines, and the term "plurality of sites" can mean up to three, four, five, six, seven, eight, nine, ten, or eleven sites, wherein the plurality of sites comprises (or consists of) up to two sites per corrugator supercilii muscle and up to one site per procerus procerus muscle for treating glabellar lines, and up to two or three sites per orbicularis oculi muscle (e.g., up to two or three sites per orbicularis oculi muscle that are located outside of the orbicularis oculi muscle) for treating lateral canthal lines. Preferably, two unit doses of the modified BoNT / A are administered to two different sites per corrugator supercilii muscle, and one unit dose is administered to one site per procerus procerus muscle for treating glabellar lines, and three unit doses of the modified BoNT / A are administered to three different sites per orbicularis oculi muscle for treating lateral canthal lines (e.g., such that eleven unit doses are administered to eleven different sites).

[0169] The facial lines to be treated can be forehead lines and lateral canthal lines, and the term "plurality of sites" can mean up to four, five, six, seven, or eight sites, wherein the plurality of sites includes (or consists of) up to two, three, four, or five sites of the frontalis muscle for treating forehead lines, and up to two or three sites of the orbicularis oculi muscle (e.g., up to two or three sites outside the orbicularis oculi muscle) for treating lateral canthal lines. Preferably, five unit doses are administered to five different sites of the frontalis muscle for treating forehead lines, and three unit doses of the modified BoNT / A are administered to three different sites of the orbicularis oculi muscle for treating lateral canthal lines (e.g., such that eight unit doses are administered to eight different sites).

[0170] The facial lines to be treated can be forehead lines and lateral canthal lines, and the term "plurality of sites" can mean up to four, five, six, seven, eight, nine, ten, or eleven sites, wherein the plurality of sites includes (or consists of) up to two, three, four, or five sites of the frontalis muscle for treating forehead lines, and up to two or three sites of each orbicularis oculi muscle (e.g., up to two or three sites outside each orbicularis oculi muscle) for treating lateral canthal lines. Preferably, five unit doses are administered to five different sites of the frontalis muscle for treating forehead lines, and three unit doses of the modified BoNT / A are administered to three different sites of each orbicularis oculi muscle for treating lateral canthal lines (e.g., such that eleven unit doses are administered to eleven different sites).

[0171] The facial lines to be treated may be frown lines, forehead lines and lateral canthal lines, and the term "multiple sites" may mean up to four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen sites, wherein the plurality of sites includes (or consists of)

[0172] - Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines,

[0173] - Up to two, three, four or five sites on the frontalis muscle for the treatment of forehead lines, and

[0174] - Up to two or three sites on the orbicularis oculi muscle (e.g., up to two or three sites on the outside of the orbicularis oculi muscle) for treatment of lateral canthal lines.

[0175] Preferred are:

[0176] - administering two unit doses of modified BoNT / A to two different sites of the corrugator supercilii muscle and one unit dose to one site of the procerus supercilii muscle for the treatment of glabellar lines,

[0177] - five unit doses injected into five different sites of the frontalis muscle for the treatment of forehead lines, and

[0178] - Three unit doses of modified BoNT / A are administered to three different sites of the orbicularis oculi muscle (e.g., the outer part thereof) for the treatment of lateral canthal lines

[0179] - (e.g., eleven unit doses are administered to eleven different sites).

[0180] The facial lines to be treated may be frown lines, forehead lines and lateral canthal lines, and the term "plurality of sites" may mean up to four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen sites, wherein the plurality of sites comprises (or consists of):

[0181] - Up to two sites on each corrugator supercilii muscle and one site on the procerus supercilii muscle for the treatment of glabellar lines,

[0182] - Up to two, three, four or five sites on the frontalis muscle for the treatment of forehead lines, and

[0183] - Up to two or three sites on each orbicularis oculi muscle (e.g., up to two or three sites on the outside of each orbicularis oculi muscle) for treatment of lateral canthal lines.

[0184] Preferred are:

[0185] - two unit doses of modified BoNT / A are applied to two different sites on each corrugator muscle and one unit dose is applied to one site on the procerus muscle for the treatment of glabellar lines,

[0186] - five unit doses injected into five different sites of the frontalis muscle for the treatment of forehead lines, and

[0187] - Three unit doses of modified BoNT / A are administered to three different locations of each orbicularis oculi muscle (e.g., its outer portion) for the treatment of lateral canthal lines

[0188] - (e.g., sixteen unit doses are administered to eleven different sites).

[0189] Therefore, the term "multiple sites" can mean up to two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen sites. Preferably, the term "multiple sites" can refer to "up to 16 sites".

[0190] The term "multiple sites" can represent two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen sites. Preferably, the term "multiple sites" can represent 16 sites.

[0191] Thus, the modified BoNT / A can be administered via intramuscular injection to up to 16 sites on the face of a subject, e.g.

[0192] - Up to four sites on the corrugator supercilii muscle (e.g., provided that the modified BoNT / A is applied to a maximum of two sites on each corrugator supercilii muscle) and one site on the procerus supercilii muscle for treatment of glabellar lines;

[0193] - Up to five sites on the frontalis muscle for the treatment of forehead lines; and

[0194] - Up to six sites of the orbicularis oculi muscle (e.g., provided that the modified BoNT / A is applied to a maximum of three sites of each orbicularis oculi muscle) for the treatment of lateral canthal wrinkles.

[0195] Multiple sites can be located on the same muscle group (e.g., left and right corrugator muscles, left and right orbicularis oculi muscles). Similarly, multiple sites can be located on the same muscle (e.g., right corrugator muscle or left corrugator muscle; or right orbicularis oculi muscle or left orbicularis oculi muscle). Similarly, multiple sites can be located on combinations of muscles and muscle groups. Thus, a personalized treatment plan can be provided.

[0196] The potency of the modified BoNT / A used according to the present invention is preferably measured by mouse LD 50 The assay was determined according to standard techniques. In the assay, 1 unit is defined as corresponding to the calculated median lethal dose (LD50) in mice. 50 ) of the modified BoNT / A. Preferably, it corresponds to the calculated median lethal intraperitoneal dose in mice.

[0197] Specifically, the amount corresponding to 1 unit of modified BoNT / A in the assay is preferably 24.04 pg.

[0198] The term "up to" when used in reference to a value (e.g., up to 3661 units) refers to up to and including that value. Thus, for example, reference to administering "up to 3661 units" of a modified BoNT / A includes administering 3661 units of a modified BoNT / A as well as administering less than 3661 units of a modified BoNT / A. As another example, reference to administering "up to 80,000 pg" of a modified BoNT / A includes administering 80,000 pg of a modified BoNT / A as well as administering less than 80,000 pg of a modified BoNT / A.

[0199] A dose of a modified BoNT / A is administered by intramuscular injection at multiple sites. A single unit dose is administered at each site. The term "administering a (single) unit dose" means administering substantially all of the (single) unit dose. Preferably, at most only (e.g., up to 1%, 0.1%, or 0.01%) of the (single) unit dose remains in the vial of reconstituted modified BoNT / A. However, preferably all of the (single) unit dose is administered. Depending on the muscle group, the (single) unit dose is typically administered to a muscle selected from the first group (described herein), 2x units are typically administered to a muscle selected from the second group (described herein), 3x units are typically administered to a muscle selected from the third group (described herein), and 5x units are typically administered to a muscle selected from the fourth group (described herein).

[0200] The unit dose can be expressed in units of modified BoNT / A. For example, 24.04 pg can correspond to 1 unit of modified BoNT / A. For example, 2404 pg of BoN / A can be expressed as 100 units of modified BoNT / A.

[0201] The unit dose may be greater than 73 units of a modified BoNT / A. The upper limit of the unit dose range may be 3,661, 3000, 2500, 2000, 1500, 1000, 500, or 100 units of a modified BoNT / A. The lower limit of the unit dose range may be 75, 100, 150, 200, 300, or 500 units of a modified BoNT / A. Preferably, the unit dose of a modified BoNT / A is greater than 73 units to 3000 units, 100 units to 2500 units, and more preferably 200 units to 2000 units.

[0202] As described above, the unit dose of the modified BoNT / A can be greater than 73 units. The upper limit of the unit dose range can be 300, 250, 200, 150, 100, or 50 units of the modified BoNT / A, preferably the upper limit is 300 units. The lower limit of the unit dose range can be 75, 100, 150, 200, 300 units, preferably the lower limit is 100 units. Preferably, the unit dose of the modified BoNT / A is 100-300 units of the modified BoNT / A, such as 150 units to 250 units.

[0203] The unit dosage of the modified BoNT / A can be selected from: 100 Units to 300 Units, 150 Units to 250 Units, and 175 Units to 225 Units.

[0204] The unit dose can be greater than 1754 pg to 8000 pg of modified BoNT / A. In other words, the unit dose can be greater than 1754 pg and up to 8000 pg of modified BoNT / A. The upper limit of the unit dose range can be 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 pg of modified BoNT / A, with a preferred upper limit of 5,000 pg. The lower limit of the unit dose range can be 1,800, 2,000, 2,500, 3000, or 4,000 pg of modified BoNT / A, with a preferred lower limit of 1,800. The unit dose of modified BoNT / A can be selected from: 1,800 to 8,000 pg, 2,000 to 6,000 pg, most preferably 3,000 to 6,000 pg. The unit dose of modified BoNT / A can be 4,500 to 5,500 pg of modified BoNT / A, such as 4,900 to 5,100 pg.

[0205] The unit dose may be greater than 1800 pg of modified BoNT / A. The unit dose may be greater than 1800 pg and up to 7000 pg of modified BoNT / A, such as greater than 1800 pg and up to 6000 pg of modified BoNT / A. The unit dose may be greater than 2000 pg and up to 5000 pg of modified BoNT / A.

[0206] The unit dose can be greater than 2555 pg. For example, the unit dose can be greater than 2555 pg and up to 8000 pg of modified BoNT / A, such as greater than 2555 pg and up to 6000 pg.

[0207] The unit dose can be 2000 pg, 2500 pg, 3000 pg, 4000 pg, 4500 pg, 5000 pg, 5500 pg, 6000 pg, 6500 pg, 7000 pg, 7500 pg, or 8000 pg of a modified BoNT / A.

[0208] More preferably, the unit dose is 2000 to 3500 pg of modified BoNT / A. For example, the unit dose can be 2250 pg to 3250 pg of modified BoNT / A. A particularly preferred unit dose can be about 2500 pg (e.g., 2500 pg ± 10%) of modified BoNT / A, for example, the unit dose can be preferably 2500 pg of modified BoNT / A. Another preferred unit dose can be about 3000 pg (e.g., 3000 pg ± 10%) of modified BoNT / A, for example, the unit dose can be 3000 pg of modified BoNT / A.

[0209] The unit dose may be 3500-5500 pg of modified BoNT / A. For example, the unit dose may be 3800 pg to 4200 pg of modified BoNT / A. The unit dose may be 4800 to 5200 pg of modified BoNT / A. A particularly preferred unit dose may be about 4000 pg (e.g., 4000 pg ± 10%) of modified BoNT / A, for example, the unit dose may be 4000 pg of modified BoNT / A. Another particularly preferred unit dose may be about 5000 pg (e.g., 5000 pg ± 10%) of modified BoNT / A, for example, the unit dose may be 5000 pg of modified BoNT / A.

[0210] A unit dose may be expressed as a "unit" or as a pg amount. A unit dose of a modified BoNT / A may also be expressed as both a unit and an amount (pg).

[0211] When treating facial lines, the modified BoNT / A can be applied to a plurality of sites selected from: up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for treating glabellar lines, up to five sites on the frontalis muscle for treating forehead lines; and up to three sites on the exterior of the orbicularis oculi muscle for treating lateral canthal lines.

[0212] The total dose administered when practicing the treatment regimen of the present invention can be up to 3661 units. In other words, the total amount of modified BoNT / A administered during a given treatment period can be up to 3661 units. The total dose can be up to 3500, 3000, 2500, 2000, 1500, or 1000 units of modified BoNT / A. Preferably, the total dose can be up to 3327.8 units of modified BoNT / A.

[0213] The total dose administered when practicing the treatment regimen of the present invention can be up to 88,000, 85,000, 80,000, 75,000, 70,000, 65,000, 60,000, 55,000, 50,000, 45,000, 40,000, 35,000, or 30,000 pg of modified BoNT / A, preferably up to 80,000 pg of modified BoNT / A. Thus, the upper limit of the total dose can be 88,000, 85,000, 80,000, 75,000, 70,000, 65,000, 60,000, 55,000, 50,000, 45,000, 40,000, 35,000, or 30,000 pg of modified BoNT / A, preferably 80,000 pg of modified BoNT / A. The lower limit of the total dose can be 25000, 20000, 15000, 10000, 5000 pg, 2000 pg, 1000 pg, 500 pg, or 50 pg of modified BoNT / A. For example, the lower limit of the total dose can be 25000, 20000, 15000, 10000, or 5000 pg of modified BoNT / A.

[0214] The total dose administered when practicing the treatment regimen of the present invention can be 7,000 pg to 8,000 pg of modified BoNT / A. Thus, 7,000 pg to 8,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 7,500 pg (e.g., 7,500 pg ± 10%) of modified BoNT / A, e.g., the total dose can be 7,500 pg of modified BoNT / A. Thus, about 7,500 pg (e.g., 7,500 pg ± 10%) of modified BoNT / A can be administered to a subject, e.g., 7,500 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein).

[0215] The total dose administered when practicing the treatment regimen of the present invention can be 10,000 pg to 20,000 pg of modified BoNT / A. Thus, 10,000 pg to 20,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). For example, the total dose can be 11,500 pg to 12,500 pg of modified BoNT / A. Thus, 11,500 pg to 12,500 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 12,000 pg (e.g., 12,000 pg ± 10%) of modified BoNT / A, for example, the total dose can be 12,000 pg of modified BoNT / A. Thus, about 12,000 pg (eg, 12,000 pg ± 10%) of a modified BoNT / A can be administered to a subject, eg, 12,000 pg of a modified BoNT / A can be administered to a subject (eg, via multiple unit doses as described herein).

[0216] For example, the total dose can be 12,000 pg to 13,000 pg of the modified BoNT / A. Thus, 12,000 pg to 13,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A, e.g., the total dose can be 12,500 pg of the modified BoNT / A. Thus, about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A can be administered to a subject, e.g., 12,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein).

[0217] The total dose administered to the subject can be 14,000 pg to 16,000 pg of the modified BoNT / A. Thus, 14,000 pg to 16,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 15,000 pg (e.g., 15,000 pg ± 10%) of the modified BoNT / A, for example, the total dose can be 15,000 pg of the modified BoNT / A. Thus, about 15,000 pg (e.g., 15,000 pg ± 10%) of the modified BoNT / A can be administered to the subject, for example, 15,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein).

[0218] The total dose administered when practicing the treatment regimen of the present invention can be 18,000 pg to 26,000 pg of modified BoNT / A. Thus, 18,000 pg to 26,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). For example, the total dose can be 19,000 pg to 21,000 pg of modified BoNT / A. Thus, 19,000 pg to 21,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, the total dose can be 20,000 pg of modified BoNT / A. Thus, about 20,000 pg (eg, 20,000 pg ± 10%) of a modified BoNT / A can be administered to a subject, eg, 20,000 pg of a modified BoNT / A can be administered to a subject (eg, via multiple unit doses as described herein).

[0219] The total dose administered when practicing the treatment regimen of the present invention can be 22,000 pg to 23,000 pg of modified BoNT / A. Thus, 22,000 pg to 23,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 22,500 pg (e.g., 22,500 pg ± 10%) of modified BoNT / A, e.g., the total dose can be 22,500 pg of modified BoNT / A. Thus, about 22,500 pg (e.g., 22,500 pg ± 10%) of modified BoNT / A can be administered to a subject, e.g., 22,500 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein).

[0220] The total dose administered to the subject can be 23,000 pg to 25,000 pg of the modified BoNT / A. Thus, 23,000 pg to 25,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A, for example, the total dose can be 24,000 pg of the modified BoNT / A. Thus, about 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A can be administered to the subject, for example, 24,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein).

[0221] The total dose administered to the subject can be 24,000 pg to 26,000 pg of the modified BoNT / A. Thus, 24,000 pg to 26,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 25,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A, e.g., the total dose can be 25,000 pg of the modified BoNT / A. Thus, about 25,000 pg (e.g., 25,000 pg ± 10%) of the modified BoNT / A can be administered to the subject, e.g., 25,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein).

[0222] The total dose administered to the subject can be 27,000 pg to 28,000 pg of the modified BoNT / A. Thus, 27,000 pg to 28,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 27,500 pg (e.g., 27,500 pg ± 10%) of the modified BoNT / A, e.g., the total dose can be 27,500 pg of the modified BoNT / A. Thus, about 27,500 pg (e.g., 27,500 pg ± 10%) of the modified BoNT / A can be administered to the subject, e.g., 27,500 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein).

[0223] The total dose administered to the subject can be 31,000 pg to 33,000 pg of the modified BoNT / A. Thus, 31,000 pg to 33,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 32,000 pg (e.g., 32,000 pg ± 10%) of the modified BoNT / A, for example, the total dose can be 32,000 pg of the modified BoNT / A. Thus, about 32,000 pg (e.g., 32,000 pg ± 10%) of the modified BoNT / A can be administered to the subject, for example, 32,000 pg of the modified BoNT / A can be administered to the subject (e.g., via multiple unit doses as described herein).

[0224] The total dose can be 35,000 pg to 37,000 pg of the modified BoNT / A. Thus, 35,000 pg to 37,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 36,000 pg (e.g., 36,000 pg ± 10%) of the modified BoNT / A, e.g., the total dose can be 36,000 pg of the modified BoNT / A. Thus, about 36,000 pg (e.g., 36,000 pg ± 10%) of the modified BoNT / A can be administered to a subject, e.g., 36,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein).

[0225] The total dose administered when practicing the treatment regimen of the present invention can be 35,000 to 45,000 pg of modified BoNT / A. Thus, 35,000 to 45,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 40,000 pg (e.g., 40,000 pg ± 10%) of modified BoNT / A, for example, the total dose can be 40,000 pg of modified BoNT / A. Thus, about 40,000 pg (e.g., 40,000 pg ± 10%) of modified BoNT / A can be administered to a subject, for example, 40,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein).

[0226] The total dose administered when practicing the treatment regimen of the present invention can be 43,000 to 44,000 pg of modified BoNT / A. Thus, 43,000 to 44,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, for example, the total dose can be 44,000 pg of modified BoNT / A. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A can be administered to a subject, for example, 44,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein).

[0227] The total dose administered when practicing the treatment regimen of the present invention can be 60,000 to 70,000 pg of modified BoNT / A. Thus, 60,000 to 70,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A preferred total dose administered when practicing the treatment regimen of the present invention can be 63,000 to 65,000 pg of modified BoNT / A. Thus, 63,000 to 65,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein). A particularly preferred total dose can be about 64,000 pg (e.g., 64,000 pg ± 10%) of modified BoNT / A, for example, a total dose can be 64,000 pg of modified BoNT / A. Thus, about 64,000 pg (eg, 64,000 pg ± 10%) of a modified BoNT / A can be administered to a subject, eg, 64,000 pg of a modified BoNT / A can be administered to a subject (eg, via a plurality of unit doses as described herein).

[0228] The total dose can be 12,000 pg to 13,000 pg of modified BoNT / A, and the unit dose can be 2000 to 3500 pg of modified BoNT / A. Thus, 12,000 pg to 13,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A.

[0229] A particularly preferred total dose can be about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A, for example, the total dose can be 12,500 pg of the modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of the modified BoNT / A. A preferred total dose is 12,500 pg of the modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of the modified BoNT / A. A more preferred total dose is 12,500 pg of the modified BoNT / A, wherein the unit dose is 2500 pg of the modified BoNT / A. Thus, 12,500 pg of the modified BoNT / A can be administered to a subject, wherein the unit dose is 2500 pg of the modified BoNT / A.

[0230] The total dose may be 14,000 pg to 16,000 pg of modified BoNT / A, and the unit dose may be 2000 to 3500 pg of modified BoNT / A. Thus, 14,000 pg to 16,000 pg of modified BoNT / A may be administered to a subject, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A. A particularly preferred total dose may be about 15,000 pg (e.g., 15,000 pg ± 10%) of modified BoNT / A, for example, the total dose may be 15,000 pg of modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A. A preferred total dose is 15,000 pg of modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A. A more preferred total dose is 15,000 pg of modified BoNT / A, wherein the unit dose is 2500 pg of modified BoNT / A. Thus, 15,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 2500 pg of modified BoNT / A.

[0231] The total dose administered when practicing the treatment regimen of the present invention can be 18,000 pg to 26,000 pg of modified BoNT / A, wherein the unit dose is 3500-5500 pg of modified BoNT / A. Thus, 18,000 pg to 26,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 3500-5500 pg of modified BoNT / A.

[0232] A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, a total dose can be 20,000 pg of modified BoNT / A, wherein the unit dose is 3500-5500 pg of modified BoNT / A. A preferred total dose is 20,000 pg of modified BoNT / A, wherein the unit dose is 3500-5500 pg of modified BoNT / A. A more preferred total dose is 20,000 pg of modified BoNT / A, wherein the unit dose is 4000 pg of modified BoNT / A. Thus, 20,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 4000 pg of modified BoNT / A.

[0233] When practicing the treatment regimen of the present invention, the total dose administered can be 23,000 pg to 25,000 pg of modified BoNT / A, wherein the unit dose is 3500-5500 pg of modified BoNT / A. Thus, 23,000 pg to 25,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 3500-5500 pg of modified BoNT / A.

[0234] A particularly preferred total dose can be about 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A, for example, a total dose can be 24,000 pg of the modified BoNT / A, wherein the unit dose is 3500-5500 pg of the modified BoNT / A. A preferred total dose is 24,000 pg of the modified BoNT / A, wherein the unit dose is 3500-5500 pg of the modified BoNT / A. A more preferred total dose is 24,000 pg of the modified BoNT / A, wherein the unit dose is 4000 pg of the modified BoNT / A. Thus, 24,000 pg of the modified BoNT / A can be administered to a subject, wherein the unit dose is 4000 pg of the modified BoNT / A.

[0235] The total dose administered when practicing the treatment regimen of the present invention can be 43,000 to 44,000 pg of modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A. Thus, 43,000 to 44,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A. A particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A, for example, the total dose can be 44,000 pg of modified BoNT / A, wherein the unit dose is 2000 to 3500 pg of modified BoNT / A. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of a modified BoNT / A can be administered to a subject, wherein the unit dose is between 2000 and 3500 pg of the modified BoNT / A. Preferably, 44,000 pg of a modified BoNT / A can be administered to a subject, wherein the unit dose is between 2000 and 3500 pg of the modified BoNT / A. For example, about 44,000 pg (e.g., 44,000 pg ± 10%) of a modified BoNT / A can be administered to a subject, wherein the unit dose is about 2500 pg (e.g., 2500 pg ± 10%) of the modified BoNT / A. More preferably, 44,000 pg of a modified BoNT / A can be administered to a subject, wherein the unit dose is 2500 pg of the modified BoNT / A.

[0236] The total dose administered when practicing the treatment regimen of the present invention can be 60,000 to 70,000 pg of modified BoNT / A, wherein the unit dose is 3500-5500 pg of modified BoNT / A. Thus, 60,000 to 70,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 3500-5500 pg of modified BoNT / A. The preferred total dose administered when practicing the treatment regimen of the present invention can be 63,000 to 65,000 pg of modified BoNT / A, wherein the unit dose is 3500-5500 pg of modified BoNT / A. Thus, 63,000 to 65,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 3500-5500 pg of modified BoNT / A. A particularly preferred total dose may be about 64,000 pg (e.g., 64,000 pg ± 10%) of the modified BoNT / A, wherein the unit dose is 3500-5500 pg of the modified BoNT / A. For example, the total dose may be 64,000 pg of the modified BoNT / A, wherein the unit dose is 3500-5500 pg of the modified BoNT / A. Thus, about 64,000 pg (e.g., 64,000 pg ± 10%) of the modified BoNT / A may be administered to the subject, wherein the unit dose is 3500-5500 pg of the modified BoNT / A. Preferably, about 64,000 pg (e.g., 64,000 pg ± 10%) of the modified BoNT / A may be administered to the subject, wherein the unit dose is about 4000 pg (e.g., 4,000 pg ± 10%) of the modified BoNT / A. More preferably, 64,000 pg of modified BoNT / A can be administered to a subject, wherein the unit dose is 4000 pg of modified BoNT / A.

[0237] The total dose may vary depending on the facial line or combination of facial lines to be treated. Examples of preferred total doses for any given facial line are discussed in more detail below.

[0238] The total dose administered when implementing the treatment regimen for glabellar lines can be up to 30,000, 25,000, 20,000, 15,000, 10,000, or 5,000 pg of modified BoNT / A, preferably up to 25,000 pg of modified BoNT / A. A particularly preferred total dose that can be administered when implementing the treatment regimen for glabellar lines can be about 12,500 pg (e.g., 12,500 pg ± 10%) of modified BoNT / A, for example, a unit dose can be 12,500 pg of modified BoNT / A. Another particularly preferred total dose that can be administered when implementing the treatment regimen for glabellar lines can be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, a unit dose can be 20,000 pg of modified BoNT / A.

[0239] The total dose administered when implementing the glabellar wrinkles treatment regimen can be 7,000 pg to 8,000 pg of the modified BoNT / A. Thus, 7,000 pg to 8,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar wrinkles. A particularly preferred total dose can be about 7,500 pg (e.g., 7,500 pg ± 10%) of the modified BoNT / A to treat glabellar wrinkles, e.g., the total dose can be 7,500 pg of the modified BoNT / A to treat glabellar wrinkles. Thus, about 7,500 pg (e.g., 7,500 pg ± 10%) of the modified BoNT / A can be administered to a subject to treat glabellar wrinkles, e.g., 7,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar wrinkles.

[0240] The total dose administered when implementing a treatment regimen for glabellar lines can be 10,000 pg to 20,000 pg of the modified BoNT / A. Thus, 10,000 pg to 20,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines. For example, the total dose can be 11,500 pg to 12,500 pg of the modified BoNT / A to treat glabellar lines. Thus, 11,500 pg to 12,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines. A particularly preferred total dose can be about 12,000 pg (e.g., 12,000 pg ± 10%) of the modified BoNT / A, for example, the total dose can be 12,000 pg of the modified BoNT / A to treat glabellar lines. Thus, about 12,000 pg (e.g., 12,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines, for example, 12,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines.

[0241] The total dose administered when implementing a treatment regimen for glabellar lines can be 12,000 pg to 13,000 pg of the modified BoNT / A. Thus, 12,000 pg to 13,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines. A particularly preferred total dose can be about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A to treat glabellar lines, e.g., the total dose can be 12,500 pg of the modified BoNT / A to treat glabellar lines. Thus, about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A can be administered to a subject to treat glabellar lines, e.g., 12,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines.

[0242] The total dose administered when implementing a treatment regimen for glabellar lines can be 18,000 pg to 26,000 pg of the modified BoNT / A. Thus, 18,000 pg to 26,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines. For example, the total dose can be 19,000 pg to 21,000 pg of the modified BoNT / A to treat glabellar lines. Thus, 19,000 pg to 21,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines. A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of the modified BoNT / A for treating glabellar lines, for example, the total dose can be 20,000 pg of the modified BoNT / A for treating glabellar lines. Thus, about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines, for example, 20,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines.

[0243] The total dose administered when implementing a treatment regimen for forehead lines can be up to 30,000, 25,000, 20,000, 15,000, 10,000, or 5,000 pg of modified BoNT / A, preferably up to 25,000 pg of modified BoNT / A.

[0244] The total dose administered when implementing a treatment regimen for forehead lines can be 12,000 pg to 13,000 pg of the modified BoNT / A. Thus, 12,000 pg to 13,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines. A particularly preferred total dose can be about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A to treat forehead lines, e.g., the total dose can be 12,500 pg of the modified BoNT / A to treat forehead lines. Thus, about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A can be administered to a subject to treat forehead lines, e.g., 12,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines.

[0245] The total dose administered when implementing a forehead wrinkle treatment regimen can be 18,000 pg to 26,000 pg of the modified BoNT / A. Thus, 18,000 pg to 26,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead wrinkles. For example, the total dose can be 19,000 pg to 21,000 pg of the modified BoNT / A to treat forehead wrinkles. Thus, 19,000 pg to 21,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead wrinkles. A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of the modified BoNT / A for treating forehead wrinkles, for example, the total dose can be 20,000 pg of the modified BoNT / A for treating forehead wrinkles. Thus, about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating forehead lines, for example, 20,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating forehead lines.

[0246] The total dose administered when implementing a lateral canthal wrinkle treatment regimen can be up to 30,000, 25,000, 20,000, 15,000, 10,000, or 5,000 pg of modified BoNT / A, preferably up to 25,000 pg of modified BoNT / A.

[0247] The total dose administered when implementing the lateral canthal wrinkle treatment regimen can be 7,000 pg to 8,000 pg of the modified BoNT / A. Thus, 7,000 pg to 8,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal wrinkles. A particularly preferred total dose can be about 7,500 pg (e.g., 7,500 pg ± 10%) of the modified BoNT / A for treating lateral canthal wrinkles, for example, the total dose can be 7,500 pg of the modified BoNT / A for treating lateral canthal wrinkles. Thus, about 7,500 pg (e.g., 7,500 pg ± 10%) of the modified BoNT / A can be administered to a subject for treating lateral canthal wrinkles, for example, 7,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal lines.

[0248] The total dose administered when implementing the lateral canthal wrinkle treatment regimen can be 10,000 pg to 20,000 pg of the modified BoNT / A. Thus, 10,000 pg to 20,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal wrinkles. For example, the total dose can be 11,500 pg to 12,500 pg of the modified BoNT / A for treating lateral canthal wrinkles. Thus, 11,500 pg to 12,500 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal wrinkles. A particularly preferred total dose can be about 12,000 pg (e.g., 12,000 pg ± 10%) of the modified BoNT / A, such as a total dose of 12,000 pg of the modified BoNT / A for treating lateral canthal wrinkles. Thus, about 12,000 pg (e.g., 12,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating lateral canthal wrinkles, for example, 12,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal wrinkles.

[0249] The total dose administered when implementing the lateral canthal wrinkle treatment regimen can be 14,000 pg to 16,000 pg of the modified BoNT / A. Thus, 14,000 pg to 16,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal lines. A particularly preferred total dose can be about 15,000 pg (e.g., 15,000 pg ± 10%) of the modified BoNT / A for treating lateral canthal wrinkles, for example, the total dose can be 15,000 pg of the modified BoNT / A for treating lateral canthal wrinkles. Thus, about 15,000 pg (e.g., 15,000 pg ± 10%) of the modified BoNT / A can be administered to a subject for treating lateral canthal wrinkles, for example, 15,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating lateral canthal wrinkles.

[0250] The total dose administered when implementing the lateral canthal wrinkle treatment regimen can be 23,000 pg to 25,000 pg of the modified BoNT / A. Thus, 23,000 pg to 25,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat lateral canthal wrinkles. A particularly preferred total dose can be about 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A for treating lateral canthal wrinkles, for example, the total dose can be 24,000 pg of the modified BoNT / A for treating lateral canthal wrinkles. Thus, about 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A can be administered to a subject for treating lateral canthal wrinkles, for example, 24,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat lateral canthal wrinkles.

[0251] The total dose administered when implementing a treatment regimen for glabellar lines and forehead lines can be 18,000 pg to 26,000 pg of the modified BoNT / A. Thus, 18,000 pg to 26,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and forehead lines. For example, the total dose can be 19,000 pg to 21,000 pg of the modified BoNT / A to treat glabellar lines and forehead lines. Thus, 19,000 pg to 21,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and forehead lines. A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A for treating glabellar lines and forehead lines, for example, a total dose can be 20,000 pg of a modified BoNT / A for treating glabellar lines and forehead lines. Thus, about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines and forehead lines, for example, 20,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines and forehead lines.

[0252] The total dose administered when implementing a treatment regimen for glabellar and forehead lines can be 24,000 pg to 26,000 pg of modified BoNT / A. Thus, 24,000 pg to 26,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar and forehead lines. A particularly preferred total dose can be about 25,000 pg (e.g., 24,000 pg ± 10%) of modified BoNT / A to treat glabellar and forehead lines, for example, a total dose can be 25,000 pg of modified BoNT / A to treat glabellar and forehead lines. Thus, about 25,000 pg (e.g., 25,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and forehead lines, for example, 25,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and forehead lines.

[0253] The total dose administered when implementing the treatment regimen for glabellar and forehead lines can be 31,000 pg to 33,000 pg of modified BoNT / A. Thus, 31,000 pg to 33,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar and forehead lines. A particularly preferred total dose can be about 32,000 pg (e.g., 32,000 pg ± 10%) of modified BoNT / A to treat glabellar and forehead lines, for example, a total dose can be 32,000 pg of modified BoNT / A to treat glabellar and forehead lines. Thus, about 32,000 pg (e.g., 32,000 pg ± 10%) of modified BoNT / A can be administered to a subject to treat glabellar lines and forehead lines, for example, 32,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and forehead lines.

[0254] The total dose administered when implementing the treatment regimen for glabellar and forehead lines can be 35,000 to 45,000 pg of modified BoNT / A. Thus, 35,000 to 45,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar and forehead lines. A particularly preferred total dose can be about 40,000 pg (e.g., 40,000 pg ± 10%) of modified BoNT / A to treat glabellar and forehead lines, for example, a total dose can be 40,000 pg of modified BoNT / A to treat glabellar and forehead lines. Thus, about 40,000 pg (e.g., 40,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and forehead lines, for example, 40,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and forehead lines.

[0255] The total dose administered when implementing the treatment regimen for glabellar lines and lateral canthal lines can be 14,000 pg to 16,000 pg of modified BoNT / A. Thus, 14,000 pg to 16,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 15,000 pg (e.g., 15,000 pg ± 10%) of modified BoNT / A for treating glabellar lines and lateral canthal lines, for example, a total dose can be 15,000 pg of modified BoNT / A for treating glabellar lines and lateral canthal lines. Thus, about 15,000 pg (e.g., 15,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and lateral canthal lines, for example, 15,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines.

[0256] The total dose administered when implementing a treatment regimen for glabellar lines and lateral canthal lines can be 18,000 pg to 26,000 pg of the modified BoNT / A. Thus, 18,000 pg to 26,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. For example, the total dose can be 19,000 pg to 21,000 pg of the modified BoNT / A to treat glabellar lines and lateral canthal lines. Thus, 19,000 pg to 21,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A for treating glabellar lines and lateral canthal lines, for example, a total dose can be 20,000 pg of a modified BoNT / A for treating glabellar lines and lateral canthal lines. Thus, about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines and lateral canthal lines, for example, 20,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines and lateral canthal lines.

[0257] The total dose administered when implementing a treatment regimen for glabellar lines and lateral canthal lines can be 22,000 pg to 23,000 pg of modified BoNT / A. Thus, 22,000 pg to 23,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 22,500 pg (e.g., 22,500 pg ± 10%) of modified BoNT / A to treat glabellar lines and lateral canthal lines, for example, a total dose can be 22,500 pg of modified BoNT / A to treat glabellar lines and lateral canthal lines. Thus, about 22,500 pg (e.g., 22,500 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and lateral canthal lines, for example, 22,500 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines.

[0258] The total dose administered when implementing a treatment regimen for glabellar lines and lateral canthal lines can be 23,000 pg to 25,000 pg of modified BoNT / A. Thus, 23,000 pg to 25,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 24,000 pg (e.g., 24,000 pg ± 10%) of modified BoNT / A to treat glabellar lines and lateral canthal lines, for example, a total dose can be 24,000 pg of modified BoNT / A to treat glabellar lines and lateral canthal lines. Thus, about 24,000 pg (e.g., 24,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and lateral canthal lines, for example, 24,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat lateral canthal lines.

[0259] The total dose administered when implementing the treatment regimen for glabellar lines and lateral canthal lines can be 27,000 pg to 28,000 pg of modified BoNT / A. Thus, 27,000 pg to 28,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 27,500 pg (e.g., 27,500 pg ± 10%) of modified BoNT / A for treating glabellar lines and lateral canthal lines, for example, a total dose can be 27,500 pg of modified BoNT / A for treating glabellar lines and lateral canthal lines. Thus, about 27,500 pg (e.g., 27,500 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and lateral canthal lines, for example, 27,500 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines and lateral canthal lines.

[0260] The total dose administered when implementing the treatment regimen for glabellar lines and lateral canthal lines can be 31,000 pg to 33,000 pg of modified BoNT / A. Thus, 31,000 pg to 33,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 32,000 pg (e.g., 32,000 pg ± 10%) of modified BoNT / A for treating glabellar lines and lateral canthal lines, for example, a total dose can be 32,000 pg of modified BoNT / A for treating glabellar lines and lateral canthal lines. Thus, about 32,000 pg (e.g., 32,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines and lateral canthal lines, for example, 32,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines and lateral canthal lines.

[0261] The total dose administered when implementing the treatment regimen for glabellar lines and lateral canthal lines can be 35,000 pg to 37,000 pg of modified BoNT / A. Thus, 35,000 pg to 37,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 36,000 pg (e.g., 36,000 pg ± 10%) of modified BoNT / A for treating glabellar lines and lateral canthal lines, for example, a total dose can be 36,000 pg of modified BoNT / A for treating glabellar lines and lateral canthal lines. Thus, about 36,000 pg (e.g., 36,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and lateral canthal lines, for example, 36,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines.

[0262] The total dose administered when implementing the treatment regimen for glabellar lines and lateral canthal lines can be 43,000 to 44,000 pg of modified BoNT / A. Thus, 43,000 to 44,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines. A particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A for treating glabellar lines and lateral canthal lines, for example, a total dose can be 44,000 pg of modified BoNT / A for treating glabellar lines and lateral canthal lines. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines and lateral canthal lines, for example, 44,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines and lateral canthal lines.

[0263] The total dose administered when implementing a treatment regimen for forehead lines and lateral canthal lines can be 18,000 pg to 26,000 pg of the modified BoNT / A. Thus, 18,000 pg to 26,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines and lateral canthal lines. For example, the total dose can be 19,000 pg to 21,000 pg of the modified BoNT / A to treat forehead lines and lateral canthal lines. Thus, 19,000 pg to 21,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines and lateral canthal lines. A particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A for treating forehead lines and lateral canthal lines, for example, a total dose can be 20,000 pg of a modified BoNT / A for treating forehead lines and lateral canthal lines. Thus, about 20,000 pg (e.g., 20,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating forehead lines and lateral canthal lines, for example, 20,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating forehead lines and lateral canthal lines.

[0264] The total dose administered when implementing a treatment regimen for forehead lines and lateral canthal lines can be 27,000 pg to 28,000 pg of modified BoNT / A. Thus, 27,000 pg to 28,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating forehead lines and lateral canthal lines. A particularly preferred total dose can be about 27,500 pg (e.g., 27,500 pg ± 10%) of modified BoNT / A for treating forehead lines and lateral canthal lines, for example, a total dose can be 27,500 pg of modified BoNT / A for treating forehead lines and lateral canthal lines. Thus, about 27,500 pg (e.g., 27,500 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat forehead lines and lateral canthal lines, for example, 27,500 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses described herein) for treating forehead lines and lateral canthal lines.

[0265] The total dose administered when implementing a treatment regimen for forehead lines and lateral canthal lines can be 31,000 pg to 33,000 pg of modified BoNT / A. Thus, 31,000 pg to 33,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines and lateral canthal lines. A particularly preferred total dose can be about 32,000 pg (e.g., 32,000 pg ± 10%) of modified BoNT / A for treating forehead lines and lateral canthal lines, for example, a total dose can be 32,000 pg of modified BoNT / A for treating forehead lines and lateral canthal lines. Thus, about 32,000 pg (e.g., 32,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat forehead lines and lateral canthal lines, for example, 32,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines and lateral canthal lines.

[0266] The total dose administered when implementing a treatment regimen for forehead lines and lateral canthal lines can be 43,000 to 44,000 pg of modified BoNT / A. Thus, 43,000 to 44,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat forehead lines and lateral canthal lines. A particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A for treating forehead lines and lateral canthal lines, for example, a total dose can be 44,000 pg of modified BoNT / A for treating forehead lines and lateral canthal lines. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat forehead lines and lateral canthal lines, for example, 44,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating forehead lines and lateral canthal lines.

[0267] The total dose administered when implementing a treatment regimen for glabellar lines, forehead lines, and lateral canthal lines can be 43,000 to 44,000 pg of modified BoNT / A. Thus, 43,000 to 44,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines, forehead lines, and lateral canthal lines. A particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines, for example, a total dose can be 44,000 pg of modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of a modified BoNT / A can be administered to a subject to treat glabellar lines, forehead lines, and lateral canthal lines, for example, 44,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines, forehead lines, and lateral canthal lines.

[0268] The total dose administered when implementing a regimen for treating glabellar lines, forehead lines, and lateral canthal lines can be 60,000 to 70,000 pg of the modified BoNT / A. Thus, 60,000 to 70,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines, forehead lines, and lateral canthal lines. The preferred total dose administered when implementing a regimen for treating glabellar lines, forehead lines, and lateral canthal lines can be 63,000 to 65,000 pg of the modified BoNT / A. Thus, 63,000 to 65,000 pg of the modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines, forehead lines, and lateral canthal lines. A particularly preferred total dose can be about 64,000 pg (e.g., 64,000 pg ± 10%) of a modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines, for example, a total dose can be 64,000 pg of a modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines. Thus, about 64,000 pg (e.g., 64,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines, forehead lines, and lateral canthal lines, for example, 64,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines, forehead lines, and lateral canthal lines.

[0269] The total dose administered when implementing a treatment regimen for glabellar lines, forehead lines, and lateral canthal lines can be 75,000 pg to 88,000 pg of modified BoNT / A. Thus, 75,000 pg to 88,000 pg of modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) to treat glabellar lines, forehead lines, and lateral canthal lines. A particularly preferred total dose can be about 80,000 pg (e.g., 80,000 pg ± 10%) of modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines, for example, a total dose can be 80,000 pg of modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines. Thus, about 80,000 pg (e.g., 80,000 pg ± 10%) of a modified BoNT / A can be administered to a subject for treating glabellar lines, forehead lines, and lateral canthal lines, for example, 80,000 pg of a modified BoNT / A can be administered to a subject (e.g., via multiple unit doses as described herein) for treating glabellar lines, forehead lines, and lateral canthal lines.

[0270] When treating glabellar lines, forehead lines, and lateral canthal lines, the total dose may be as high as 88,000 pg (eg, up to 80,000 pg).

[0271] The skilled artisan will take into account whether the subject has recently received (or has subsequently received) additional treatment with a Clostridial neurotoxin (e.g., BoNT), for example as part of a cosmetic treatment or treatment for a different indication. Using routine techniques in the art, the skilled artisan will adjust the present treatment regimen accordingly.

[0272] According to the invention, administration to multiple muscles is preferably performed during the same treatment session.

[0273] Compared with unmodified BoNT / A (e.g. ). The modified BoNT / A of the present invention preferably has a longer duration of action than the duration of treatment with an unmodified BoNT / A. The term "the duration of treatment of facial wrinkles is longer than the duration of treatment with an unmodified BoNT / A" may mean that facial wrinkles are reduced for a longer period of time after administration of a modified BoNT / A of the present invention compared to administration of an unmodified BoNT / A. The duration of action may be at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25×. The duration of action of a modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least: 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In certain embodiments, the duration of action may be greater than 9.0 months. Preferably, the duration of action may be greater than 9.0 months. The reduction may be determined by comparison to an equivalent control subject exhibiting equivalent symptoms, which equivalent symptoms have been treated with an unmodified BoNT / A. A subject treated with a modified BoNT / A according to the present invention can exhibit an equivalent improvement in one or more symptoms, at least 5%, 10%, 25%, or 50% improvement compared to the severity of one or more symptoms prior to treatment with the modified BoNT / A, within a time period that is substantially the same (e.g., the same) as the severity of one or more symptoms in a control subject prior to treatment with the unmodified BoNT / A. The unmodified BoNT / A is preferably SEQ ID NO: 2 in a two-stranded form. In other words, the unmodified BoNT / A described herein is preferably a two-stranded form of SEQ ID NO: 2.

[0274] Treatment can be repeated at appropriate time intervals after administration of the modified BoNT / A. ), so the interval between subsequent administrations is longer than with unmodified BoNT / A (e.g. ) is appropriately longer when treating a subject. A modified BoNT / A according to the present invention can be re-administered to a subject at least 18, 20, 25, or 30 weeks after a previous administration. For example, a modified BoNT / A according to the present invention can be re-administered to a subject at least 18-45 weeks, preferably 20-35 weeks, after a previous administration.

[0275] As used herein, a "subject" can be a mammal, such as a human or other mammal. Preferably, a "subject" refers to a human subject. The subject is preferably a subject that does not have a disease state associated with the muscle to which the modified BoNT / A is administered. For example, preferably, the subject does not have blepharospasm or hemifacial spasm (e.g., classic hemifacial spasm). Particularly preferably, the subject does not have blepharospasm.

[0276] A subject treated in accordance with the present invention may be a subject that is not suitable for treatment with an unmodified BoNT / A and / or with another clostridial neurotoxin. The subject may be a subject that is resistant to treatment with an unmodified BoNT / A and / or another clostridial neurotoxin. Resistance may result from an immune response in the subject to the clostridial neurotoxin, including the production of anti-clostridial neurotoxin antibodies.

[0277] The terms "treat" or "treating" refer to cosmetic treatments of facial lines, including preventative treatments (e.g., preventing the appearance of facial lines) as well as corrective treatments (treating a subject who already has facial lines). Thus, any method, modified BoNT / A employed, or use described herein may be appropriately described as "a cosmetic method of using the modified BoNT / A to treat the facial lines." Additionally or alternatively, any method, modified BoNT / A employed, or use described herein may be appropriately described as "a non-therapeutic use of the modified BoNT / A to treat the facial lines."

[0278] Preferably, "treat" or "treating" as used herein refers to corrective treatment. The term "treat" or "treating" as used herein refers to facial lines and / or symptoms thereof. The treatments described herein may reduce the level of glabellar lines by at least 5%, 10%, 25%, or 50% relative to the level of glabellar lines before treatment. The treatments described herein may reduce the level of forehead lines by at least 5%, 10%, 25%, or 50% relative to the level of forehead lines before treatment. The treatments described herein may reduce the level of lateral canthal lines by at least 5%, 10%, 25%, or 50% relative to the level of lateral canthal lines before treatment. The treatments described herein may reduce the level of glabellar lines and lateral canthal lines by at least 5%, 10%, 25%, or 50% relative to the level of glabellar lines and lateral canthal lines before treatment. The treatments described herein may reduce the level of glabellar lines and forehead lines by at least 5%, 10%, 25%, or 50% relative to the level of glabellar lines and forehead lines before treatment. Treatments described herein may reduce the level of lateral canthal lines and forehead lines by at least 5%, 10%, 25%, or 50% relative to the level of lateral canthal lines and forehead lines before treatment. Treatments described herein may reduce the level of glabellar lines, lateral canthal lines, and forehead lines by at least 5%, 10%, 25%, or 50% relative to the level of glabellar lines, lateral canthal lines, and forehead lines before treatment.

[0279] BoNT / A is an example of a clostridial neurotoxin produced by Clostridium bacteria. Other examples of such clostridial neurotoxins include toxins produced by Clostridium tetani (TeNT) and Clostridium botulinum (BoNT) serotypes BG and X (see WO2018 / 009903 A2), as well as toxins produced by Clostridium baratii and Clostridium butyricum. The neurotoxins are highly potent and specific, toxic to neurons and other cells to which they are delivered. Clostridial toxins are among the strongest toxins known. For example, the median lethal dose (LD50) of botulinum neurotoxin in mice is 1.5 mmol / l. 50 ) values ​​range from 0.5 to 5 ng / kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, whereas tetanus toxin acts on the central nervous system.

[0280] In nature, clostridial neurotoxins, including BoNT / A, are synthesized as single-chain polypeptides that are modified post-translationally by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, often referred to as the activation site (e.g., the activation loop), which is located between the cysteine ​​residues that provide the interchain disulfide bond. This two-chain form is the active form of the toxin. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain contains the N-terminal translocation element (H N domain) and the C-terminal targeting component (H C domain). The cleavage site is located between the L chain and the translocation domain component. C domain binds to its target neuron and after internalization of the bound toxin into the cell via endocytosis, H N The domain translocates the L chain across the endosomal membrane and into the cytosol, while the L chain provides the protease function (also known as non-cytotoxic protease).

[0281] Non-cytotoxic proteases act by proteolytic cleavage of intracellular trafficking proteins known as SNARE proteins, such as SNAP-25, VAMP or Syntaxin - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE is derived from the term soluble NSF attachment receptor ( S oule N SF A ttachment R eceptor, where NSF stands for N-ethylmaleimide sensitive factor ( N -ethylmaleimide- S ensitive- F acor). SNARE proteins are essential for intracellular vesicle fusion and therefore for secretion of molecules from cells via vesicle transport. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cells, the non-cytotoxic protease is able to inhibit cellular secretion of the target cells. The L-chain protease of Clostridial toxins is a non-cytotoxic protease that cleaves SNARE proteins.

[0282] Given the ubiquity of SNARE proteins, clostridial neurotoxins such as botulinum toxin have been successfully used in a variety of therapeutic applications.

[0283] For more detailed information on the genetic basis of toxin production by Clostridium botulinum and Clostridium tetani, see Henderson et al. (1997), Clostridia: Molecular Biology and Pathogenesis, Academic Press, USA.

[0284] As mentioned above, the clostridial neurotoxin is composed of two polypeptide chains, the heavy chain (H chain) has a molecular weight of about 100 kDa, and the light chain (L chain) has a molecular weight of about 50 kDa. The H chain contains the C-terminal targeting component (receptor binding domain or H C domain) and the N-terminal translocation component (H N domain).

[0285] Clostridial neurotoxin domains are described in more detail below.

[0286] Examples of L chain reference sequences include:

[0287] Botulinum neurotoxin type A: amino acid residues 1-448

[0288] Botulinum neurotoxin type B: amino acid residues 1-440

[0289] The above reference sequences should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

[0290] Botulinum neurotoxin type A: amino acid residues M1-K448

[0291] Botulinum neurotoxin type B: amino acid residues M1-K441

[0292] The translocation domain is a fragment of the clostridial neurotoxin H chain corresponding approximately to the amino-terminal half of the H chain, or a domain corresponding to this fragment in the intact H chain.

[0293] Examples of reference translocation domains include:

[0294] Botulinum neurotoxin type A - amino acid residues (449-871)

[0295] Botulinum neurotoxin type B - amino acid residues (441-858)

[0296] The above reference sequences should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (incorporated herein by reference) cites a slightly different Clostridium sequence:

[0297] Botulinum neurotoxin type A - amino acid residues (A449-K871)

[0298] Botulinum neurotoxin type B - amino acid residues (A442-S858)

[0299] In the context of the present invention, various BoNT / AH comprising a translocation domain N Regions may be used in aspects of the invention. N The region is approximately 410-430 amino acids long and contains the translocation domain. N The entire length of the region is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment can include a BoNT / AH comprising a translocation domain. N The translocation domain may be, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids in length. Other aspects of this embodiment may include a BoNT / AH comprising a translocation domain. N The translocation domain may be, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids in length.

[0300] Terminology N Covers naturally occurring BoNT / AH N portions, and modified BoNT / AHs having amino acid sequences not found in nature and / or synthetic amino acid residues N Preferably, the modified BoNT / AH N Some still show the above-mentioned translocation function.

[0301] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include:

[0302] BoNT / A——N872-L1296

[0303] BoNT / B——E859-E1291

[0304] Clostridial neurotoxins (such as BoNT) have a ~50 kDa H C The domain contains two distinct structural features, called H CC and H CN domains, each structural feature is usually ~25 kDa. The amino acid residues involved in receptor binding are thought to be mainly located in the H CC Domain. H of natural Clostridial neurotoxin CA domain may contain about 400-440 amino acid residues. This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4:788-792; Herreros J (2000) Biochem. J. 347:199-204; Halpern J (1993) J. Biol. Chem. 268:15, pp. 11188-11192; Rummel A (2007) PNAS 104:359-364; Lacey DB (1998) Nat. Struct. Biol. 5:898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25:90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7:1751-1759; and Rummel A(2004) Mol. Microbiol. 51(3), 631-643.

[0305] H CN Examples of domains (references) include:

[0306] Botulinum neurotoxin type A - amino acid residues (872-1110)

[0307] Botulinum neurotoxin type B - amino acid residues (859-1097)

[0308] The above sequence positions may vary slightly depending on the serotype / subtype, (reference) H CN Further examples of domains include:

[0309] Botulinum neurotoxin type A - amino acid residues (874-1110)

[0310] Botulinum neurotoxin type B - amino acid residues (861-1097)

[0311] H CC Examples of domains (references) include:

[0312] Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

[0313] Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

[0314] WO 2017 / 191315 A1 (incorporated herein by reference) teaches modified BoNT / A (e.g., "chimeric clostridial neurotoxins") and methods for their preparation and manufacture. Thus, a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (BoNT / AH) for use in the present invention may be a chimeric clostridial neurotoxin. N ) and the BoNT / B receptor binding domain (H C The modified BoNT / A having a 347 amino acid sequence (A) or a 347 amino acid sequence (B) may be the modified BoNT / A taught in WO 2017 / 191315 A1.

[0315] The modified BoNT / A may be referred to as a "chimeric clostridial neurotoxin". As used herein, the term "chimeric clostridial neurotoxin" or "chimeric neurotoxin" refers to a clostridial neurotoxin light chain and a translocation domain (H domain) from a first clostridial neurotoxin serotype. N domain) and a receptor binding domain (H C In particular, the modified BoNT / A (e.g., chimeric clostridial neurotoxin) for use in the present invention comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C Domain). BoNT / A LH of a modified BoNT / A (e.g., a chimeric Clostridial neurotoxin) N Domains and BoNT / BH C The domains are covalently linked. The modified BoNT / A (e.g., chimeric clostridial neurotoxin) of the present invention can be referred to as a chimeric botulinum neurotoxin. The chimeric clostridial neurotoxin is also referred to herein as a "BoNT / AB," "mrBoNT / AB," or "BoNT / AB chimera."

[0316] L chain and H N The domains (optionally including a complete or partial activation loop, e.g., a complete activation loop when the modified BoNT / A is in single-chain form, a cleaved / partial activation loop when the modified BoNT / A is in two-chain form) may be collectively referred to as LH N domain. Therefore, LH N The domain does not further include H C Structural domain.

[0317] The modified BoNT / A may consist essentially of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H Cdomain).

[0318] The term "consisting essentially of" means that the modified BoNT / A does not further comprise one or more amino acid residues that confer additional functionality to the polypeptide (e.g., when administered to a subject). In other words, "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C The polypeptide composed of a botulinum toxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C The amino acid residues of the polypeptide (or domain) are the same), but the one or more other amino acid residues do not confer additional functions to the polypeptide (e.g., when administered to a subject). The additional functions may include enzymatic activity, binding activity and / or any physiological activity.

[0319] The modified BoNT / A may also include non-Clostridial neurotoxin sequences in addition to any Clostridial neurotoxin sequence, as long as the non-Clostridial neurotoxin sequence does not destroy the ability of the modified BoNT / A (e.g., chimeric Clostridial neurotoxin) to achieve its therapeutic effect. Preferably, the non-Clostridial neurotoxin sequence is not a sequence with catalytic activity (e.g., enzymatic activity). In one embodiment, the modified BoNT / A of the present invention does not include a non-Clostridial catalytically active domain. In one embodiment, the modified BoNT / A does not include a further catalytically active domain. In one embodiment, the non-Clostridial sequence is not a sequence that binds to a cell receptor. In other words, in one embodiment, the non-Clostridial sequence is not a ligand for a cell receptor. The cell receptor may be a protein cell receptor, such as an integral membrane protein. Examples of cell receptors can be found in the IUPHAR Guide to Pharmacology Database, Version 2019.4, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-Clostridial neurotoxin sequence may include a tag that facilitates purification, such as a His tag. In one embodiment, a modified BoNT / A of the invention does not comprise a tag or a site for adding a tag, such as a sortase acceptor or donor site.

[0320] Preferably, the modified BoNT / A may be composed of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0321] The modified BoNT / A comprises a light chain that is capable of exhibiting non-cytotoxic protease activity and is capable of cleaving SNARE proteins in the cytosol of target neurons. Cell-based and in vivo assays can be used to determine whether a Clostridial neurotoxin comprising an L chain and a functional cell binding and translocation domain has non-cytotoxic protease activity. Assays such as the toe abduction score (DAS) assay, dorsal root ganglion (DRG) assay, spinal cord neuron (SCN) assay, and mouse phrenic nerve hemidiaphragm (PNHD) assay are routinely used in the art. Suitable assays for determining non-cytotoxic protease activity may be those described in Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al. (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0322] When administered to a subject, the modified BoNT / A is preferably in its active two-chain form, wherein the light chain and the heavy chain are linked together by a disulfide bond. When a BoNT / A (e.g., a modified BoNT / A) is defined herein by a polypeptide sequence (SEQ ID NO), the L chain portion of the sequence (SEQ ID NO) can constitute the first chain of the two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A), and the H chain portion can constitute the first chain of the two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A). N and H C The domains together may constitute the second chain of a two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A), wherein the first chain and the second chain are linked together by a disulfide bond. The skilled artisan will appreciate that the protease may cleave at one or more positions within the activation loop of the clostridial neurotoxin (e.g., a modified BoNT / A), preferably at two positions within the activation loop. When cleavage occurs at more than one position (preferably two positions) within the activation loop, a small fragment of the C-terminal L chain portion of the sequence may not be present in the two-chain clostridial neurotoxin sequence (e.g., a two-chain modified BoNT / A). In view of this, the sequence of a two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A) may differ slightly from the sequence of the corresponding single-chain clostridial neurotoxin (e.g., a single-chain modified BoNT / A). The small fragment may be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, the small fragment that is not present in the C-terminal L chain portion of the sequence may be SEQ ID NO: 9 or 10.

[0323] Most preferably, the modified BoNT / A for use in the present invention may comprise a BoNT / A light chain and a translocation domain (BoNT / ALH N domain) and BoNT / BH C Domain. BoNT / A LH NDomains and BoNT / BH C The modified BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0324] LH N The C-terminal amino acid residue of the domain may correspond to the LH that separates BoNT / A N and H C Domain 3 10 The first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain may correspond to the LH in the BoNT / B N and H C Domain 3 10 The second amino acid residue of the helix.

[0325] An example of a BoNT / A polypeptide sequence is shown in SEQ ID NO:2.

[0326] An example of a BoNT / B polypeptide sequence is SEQ ID NO: 8 (UniProt Accession No. B1 INP5).

[0327] LH that separates BoNT / A N and H C Domain 3 10 The first amino acid residue of the helix is ​​the N and H C Domain 3 10 N-terminal residue of the helix.

[0328] LH that separates BoNT / B N and H C Domain 3 10 The second amino acid residue of the helix is ​​the N and H C Domain 3 10 The amino acid residue following the N-terminal residue of the helix.

[0329] "3 10 A helix is ​​a secondary structure found in proteins and peptides, along with alpha helices, beta sheets, and reverse turns. 10 The amino acids in the helix are arranged in a right-handed helical structure, in which each complete turn is completed by three residues and ten atoms separated by intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., there are three residues per turn in the helix), and the translation along the helical axis is (=0.2 nm), and there are 10 atoms in the ring formed by hydrogen bonding. Most importantly, the NH group of the amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before; this repeated i+3→i hydrogen bond defines the 3 10 Spiral. 3 10 The helix is ​​a standard concept in structural biology and is familiar to those skilled in the art.

[0330] This 3 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of the four residues. N and H C Domain 3 10 The "helix" is composed of these six residues.

[0331] Through structural analysis and sequence alignment, a 3 10 spiral, which converts LH N and H C The 3 domains are separated. 10 The N-terminal end of the helix (i.e., LH N The C-terminal part of the domain is surrounded by an α-helix, and the C-terminal (i.e., H C The N-terminal part of the domain) is surrounded by β-strands. 10 The first (N-terminal) residue (cap or transition residue) of a helix also corresponds to the C-terminal residue of the alpha helix.

[0332] Separation of LH N and H C Domain 3 10 Helices can be determined, for example, by publicly available botulinum neurotoxin crystal structures, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=1EPW) representing botulinum neurotoxins A1 and B1, respectively.

[0333] Publicly available computer modeling and alignment tools can also be used to identify LH compartments in other neurotoxins. N and H C Domain 3 10The positions of the helices are determined by, for example, the homology modeling servers LOOPP (Learn, Observe and Export Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / Mock Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and many other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). In particular, “H N / H CN “The region surrounding the junction is likely to be highly structurally conserved, making it an ideal region for superimposing different serotypes.

[0334] For example, the following method can be used to determine the presence of this 3 10 The sequence of the spiral:

[0335] 1. The structural homology modeling tool LOOP (http: / / loopp.org) can be used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb);

[0336] 2. The structure (pdb) file obtained in this way can be edited to contain only H CN The N-terminus of the domain and the approximately 80 residues preceding it (belonging to the H N domain), thereby retaining the highly conserved "H N / H CN "area;

[0337] 3. The protein overlay server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) can be used to overlay each serotype onto the 3BTA.pdb structure;

[0338] 4. You can check the superimposed pdb files to locate the H of BoNT / A1 C 3 at the beginning of the domain 10 helix and can then identify the corresponding residues in the other serotype.

[0339] 5. Other BoNT serotype sequences can be aligned with Clustal Omega to check whether the corresponding residues are correct.

[0340] LH N , H C and 3 10 An example of a helical domain is shown below:

[0341]

[0342]

[0343] Structural analysis and sequence alignment revealed that among all botulinum and tetanus neurotoxins, the LH N and H C Domain 3 10 The β-strands following the helix are all conserved structures and separate LH N and H C Domain 3 10 The first residue of the helix begins at residue 8 (eg, for BoNT / A1, at residue 879).

[0344] The BoNT / AB chimera can contain H from BoNT / B C Domain covalently linked to LH from BoNT / A N domain, in which LH N The C-terminal amino acid residue of the domain corresponds to the H C The eighth amino acid residue from the N-terminus of the β-strand of the start (N-terminus) of the domain, and wherein H C The N-terminal amino acid residue of the domain corresponds to the H C The N-terminal seventh amino acid residue of the β strand from the start of the domain (N-terminus).

[0345] The BoNT / AB chimera can contain H from BoNT / B C Domain covalently linked to LH from BoNT / A N domain, in which LH N The C-terminal amino acid residue of the domain corresponds to the LH N The C-terminal amino acid residue of the alpha helix at the end of the domain (C-terminus), and wherein H C The N-terminal amino acid residues of the domain correspond to the LH residues located immediately adjacent to the BoNT / B N The amino acid residue at the C-terminal end of the alpha helix at the end of the domain (C-terminus).

[0346] The design principle of BoNT / AB chimeras is to ensure that the secondary structure is as intact as possible, thereby minimizing any changes to the tertiary structure and the function of each domain. Without being bound by theory, it is hypothesized that by not disrupting the 3 10 The four central amino acid residues of the helix can ensure that the modified BoNT / A (eg, chimeric neurotoxin) has an optimal conformation, thereby enabling the modified BoNT / A (eg, chimeric neurotoxin) to fully exert its function.

[0347] In fact, surprisingly, only 3 10 The first amino acid residue of the helix and the 3 10 The second amino acid residue after the helix can not only produce a soluble, functional BoNT / AB chimera, but also produce properties that are superior to other BoNT / AB chimeras, particularly improved potency, improved safety ratio, and / or extended duration of action (as well as improved safety ratio and / or increased duration of action compared to an unmodified BoNT / A - e.g., SEQ ID NO: 2, such as SEQ ID NO: 2 in a double-chain form).

[0348] BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C The domain can be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C Domains or derivatives thereof, including but not limited to those described below. Modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / BH C The domain or derivative may contain one or more components that are compatible with a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C The native (unmodified) form of the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH chain may contain one or more amino acids that have been modified compared to the native (unmodified) form of the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH chain. C For example, relative to a native (unmodified) BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C Domain sequences, modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C The domains may have modified amino acid sequences in one or more domains. Such modifications may modify aspects of their function, such as biological activity or persistence. Thus, in one embodiment, a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH CThe domain is a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C domain, or a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C Domain derivatives.

[0349] Modified BoNT / BH C The domain may have one or more modifications to alter binding to target neural cells, such as native (unmodified) BoNT / BH C Compared with the other domains, they provide higher or lower affinity binding. C Such modifications in the domain may include modifying H C The invention relates to a modified neurotoxin that is directed to a ganglioside binding site of a target neuron or a protein (e.g., synaptotagmin) binding site of a target neuron, thereby altering the binding to the target neuron's ganglioside receptor and / or protein receptor. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0350] The modified light chain may have one or more modifications in its amino acid sequence, such as modifications in the substrate binding or catalytic domains, which may alter or modify the SNARE protein specificity of the modified light chain, preferably provided that the modification does not catalytically inactivate the light chain. Examples of such modified neurotoxins are described in WO 2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0351] LH of BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 2, or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 2, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:2.

[0352] BoNT / B H C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 8, or a polypeptide sequence having at least 70% sequence identity thereto. CThe domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:8.

[0353] Preferably, the BoNT / AB chimera comprises BoNT / A1 LH N Domains and BoNT / B1 H C More preferably, LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), and the H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO: 8).

[0354] Most preferably, BoNT / BH C The domain also contains H CC at least one amino acid residue substitution, insertion, indel, or deletion in a subdomain of the BoNT / B sequence, compared to a native BoNT / B sequence. CC The subdomain has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human SytII. WO 2013 / 180799 and WO 2016 / 154534 (both incorporated herein by reference) disclose BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains.

[0355] H CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may include substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0356] H CCSuitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may also include a combination of two substitution mutations selected from the following: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y or E1191V and W1178Q.

[0357] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may also include a combination of three substitution mutations, namely E1191M, S1199W and W1178Q.

[0358] BoNT / BH CC Amino acid residue substitutions, insertions, insertions / deletions, or deletions in a subdomain include a combination of two substitution mutations, namely, E1191M and S1199Y. Such modifications are present in, for example, modified BoNT / As (e.g., BoNT / AB chimeras) of SEQ ID NO: 5 and SEQ ID NO: 6. E1191M can correspond to position 1204 of SEQ ID NO: 6, and S1199Y can correspond to position 1212. Thus, SEQ ID NO: 6 can include 1204M and 1212Y.

[0359] The modification can be a modification compared to the unmodified BoNT / B set forth in SEQ ID NO:8, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:8. Since the presence of a methionine residue at position 1 of SEQ ID NO:8 (and the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, the skilled artisan will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, when SEQ ID NO:8 includes a methionine, the position numbering will be as defined above (e.g., E1191 will be E1191 of SEQ ID NO:8). Alternatively, when a methionine is not present in SEQ ID NO:8, the amino acid residue numbering should be modified by -1 (e.g., E1191 will be E1190 of SEQ ID NO:8). Thus, the initial methionine amino acid residue of a modified BoNT / A polypeptide sequence can be optional or absent. Similar considerations apply when a methionine at position 1 of other polypeptide sequences described herein is present / absent, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0360] The modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. Preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3-7.

[0361] Preferably, the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) SEQ ID NO: 6.

[0362] The term "deletion" as used herein refers to the removal of one or more amino acid residues in a polypeptide, while one or more amino acid residues at the deletion site are not replaced. Thus, when an amino acid residue is deleted from a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x-1 amino acid residues.

[0363] As used herein, the term "indel" refers to the deletion of one or more amino acid residues in a polypeptide, as well as the insertion of a different number of amino acid residues (more or fewer amino acid residues) than the number of amino acid residues deleted at the deletion site. Thus, for an indel that deletes two amino acid residues from a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x-1 amino acid residues or x+≥1 amino acid residues. Insertions and deletions can be performed in any order, consecutively, or simultaneously.

[0364] The term "substitution" as used herein refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same position. Thus, for a substitution of a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0365] The term "insertion" as used herein refers to the addition of one or more amino acid residues of a polypeptide at an insertion site without deleting one or more amino acid residues of a polypeptide. Thus, when an amino acid residue is inserted into a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x+1 amino acid residues.

[0366] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modifying a DNA sequence encoding a BoNT / A (e.g., encoding an unmodified BoNT / A). This can be accomplished using standard molecular cloning techniques, such as by site-directed mutagenesis, in which short strands of DNA (oligonucleotides) encoding the desired amino acids are used to replace the original coding sequence using a polymerase, or by inserting / deleting portions of a gene using a variety of enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized. In general, the modification can be made by modifying a nucleic acid encoding a native clostridial neurotoxin (or a portion thereof) such that the modified BoNT / A (or a portion thereof) encoded by the nucleic acid comprises the modification. Alternatively, a nucleic acid encoding a modified clostridial neurotoxin (or a portion thereof) comprising the modification can be synthesized.

[0367] When the polypeptide sequence of the modified BoNT / A described herein comprises a tag (e.g., a His tag for purification), the tag is optional. Preferably, the tag is removed prior to use of the modified BoNT / A according to the present invention.

[0368] As described above, the modified BoNT / A described herein have enhanced tissue retention properties, which also provide enhanced efficacy and / or duration of action and can allow for increased dosage without any additional negative effects. One way to define these favorable properties is the safety ratio of the modified BoNT / A. In this regard, the adverse effects of Clostridial toxins (caused by diffusion of the toxin from the site of administration) can be experimentally assessed by measuring the percentage of weight loss in relevant animal models (e.g., mice, where weight loss is detected within seven days after administration). Conversely, the desired on-target effects of Clostridial toxins can be experimentally assessed by the digital abduction score (DAS) assay, a measure of muscle paralysis. The DAS assay can be performed by injecting 20 μl of Clostridial toxin (formulated in gelatin phosphate buffer) into the gastrocnemius / soleus complex of mice and then assessing the digital abduction score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which the mice extend their hind limbs and abduct their hind fingers. After injection of the clostridial toxin, varying degrees of digital abduction were scored on a five-point scale (0 = normal to 4 = reduced digital abduction and maximal leg extension).

[0369] The safety ratio of a neurotoxin (e.g., a modified BoNT / A of the present invention (or an unmodified BoNT / A for comparison)) can then be expressed as the ratio between the amount of toxin required to achieve a 10% decrease in body weight (measured at peak effect within the first seven days after dosing in mice) and the amount of neurotoxin required for a DAS score of 2. Thus, a high safety ratio score is desirable and indicates that the neurotoxin is able to effectively paralyze the target muscle with few adverse off-target effects. The safety ratio of the modified BoNT / A of the present invention is higher than the safety ratio of the equivalent unmodified (natural) BoNT / A.

[0370] A high safety ratio is particularly advantageous in therapy because it represents an increase in the therapeutic index. In other words, this means that reduced doses can be used and / or increased doses can be used without any additional (e.g., deleterious) effects compared to alternative clostridial neurotoxin therapies. The possibility to use higher doses of the neurotoxin without additional effects is particularly advantageous because higher doses generally result in a longer duration of action of the neurotoxin.

[0371] The potency of a modified BoNT / A can be expressed as the minimum dose of the neurotoxin that results in a given DAS score when administered to the gastrocnemius / soleus complex of mice, e.g., a DAS score of 2 (ED 50 dose) or a DAS score of 4. The potency of the modified BoNT / A can also be expressed as the EC in a cellular assay measuring SNARE cleavage by neurotoxins. 50Dose, e.g. EC in a cell-based assay measuring cleavage of SNAP25 by modified BoNT / A 50 dose.

[0372] The duration of action of a modified BoNT / A can be expressed as the time required to restore a DAS score of 0 following administration of a given dose of neurotoxin (e.g., the minimum dose of neurotoxin that results in a DAS score of 4) to the gastrocnemius / soleus complex of mice.

[0373] Thus, in one embodiment, a modified BoNT / A of the invention has a safety ratio greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), wherein the safety ratio is calculated as follows: - the toxin dose required for a 10% change in body weight (pg / mouse) divided by the DAS ED 50 (pg / mouse) [ED 50 =dose required to produce a DAS score of 2]. For example, the modified BoNT / A can have a safety ratio of at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

[0374] In one embodiment, a modified BoNT / A of the invention has a safety ratio of at least 10. In one embodiment, a modified BoNT / A of the invention has a safety ratio of at least 15.

[0375] Preferably, the modified BoNT / A has a safety ratio of at least 10 (eg, a safety ratio of 10), more preferably at least 12 or 13 (eg, 14-15).

[0376] The safety ratio of the modified BoNT / A can be greater than 7 to 50, such as 8-45, 10-20, or 12-15.

[0377] In use, the modified BoNT / A of the present invention is in a double-chain form.

[0378] The modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. Preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3-7. Among the modified BoNT / A neurotoxins (e.g., chimeric clostridial neurotoxins), SEQ ID NO: 6 is preferred.

[0379] Thus, preferably, the modified BoNT / A (e.g., chimeric Clostridial neurotoxin) comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. More preferably, the polypeptide sequence has at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A (e.g., chimeric Clostridial neurotoxin) for use in the present invention can comprise (more preferably consist of) SEQ ID NO: 6.

[0380] The double-chain modified BoNT / A of the present invention may include an L chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7, constituting the first chain of the double-chain modified BoNT / A, and may include an H chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7. N and H C The structural domains together constitute the second chain of the double-chain modified BoNT / A, wherein the first chain and the second chain are connected together by a disulfide bond.

[0381] When cleavage occurs at more than one position (preferably two positions) within the activation loop of a modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7, then small fragments of the C-terminal L chain portion of a sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7) may differ slightly from the sequence of a corresponding single-chain modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7. A small fragment may be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain clostridial neurotoxin, the small fragment of the C-terminal L chain portion of the sequence that is deleted may be SEQ ID NO: 9 or 10.

[0382] Preferably, the double-chain modified BoNT / A of the present invention may include an L chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6, constituting the first chain of the double-chain modified BoNT / A, and may include an H chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6. N and H C The structural domains together constitute the second chain of the two-chain modified BoNT / A, wherein the first chain and the second chain are linked together by a disulfide bond.

[0383] When cleavage occurs at more than one position (preferably two positions) within the activation loop of a modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6, then small fragments of the C-terminal L chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a double-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6) may differ slightly from the sequence of a corresponding single-chain modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6, and the small fragment may be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a double-chain modified BoNT / A, the small fragment of the C-terminal L chain portion of the sequence that is deleted may be SEQ ID NO: 9 or 10.

[0384] In particularly preferred embodiments, the two-chain modified BoNT / A comprises a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising (or consisting of) a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. More preferably, the two-chain modified BoNT / A comprises a light chain comprising SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising (or consisting of) SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. Even more preferably, the two-chain modified BoNT / A comprises a light chain having SEQ ID NO: 11 and a heavy chain having SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by and / or between cysteine ​​residue 429 of SEQ ID NO: 11 or 12 and cysteine ​​residue 6 of SEQ ID NO: 13.

[0385] In preferred embodiments, the modified BoNT / A (e.g., chimeric clostridial neurotoxin) of the invention does not comprise a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) other than the light and heavy chains. For example, in one embodiment, a modified BoNT / A (e.g., chimeric clostridial neurotoxin) may not comprise a covalently or non-covalently associated therapeutic or diagnostic agent. Thus, the modified BoNT / A (e.g., chimeric clostridial neurotoxin) of the invention preferably does not serve as a delivery vehicle for further therapeutic or diagnostic agents.

[0386]

[00136] In embodiments where a modified BoNT / A (e.g., a chimeric Clostridial neurotoxin) described herein has a tag (e.g., a His tag) and / or a linker for purification, the tag and / or linker is optional.

[0387] The modified BoNT / A is preferably in a non-complexed form (i.e., may not contain complexing proteins present in a naturally occurring clostridial neurotoxin complex (e.g., a BoNT / A complex)). Examples of such complexing proteins include neurotoxin associated protein (NAP) and non-toxic non-hemagglutinin component (NTNH). However, the modified BoNT / A is preferably a recombinant modified BoNT / A. The modified BoNT / A of the present invention can be produced using recombinant nucleic acid technology. Thus, in one embodiment, the modified BoNT / A (as described herein) is a recombinant modified BoNT / A (e.g., a chimeric clostridial neurotoxin).

[0388] In one embodiment, a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a modified BoNT / A is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0389] In a preferred embodiment, the vector has a promoter selected from:

[0390]

[0391] In another preferred embodiment, the vector has a promoter selected from the following:

[0392]

[0393] Nucleic acid molecules can be prepared using any suitable method known in the art. Thus, nucleic acid molecules can be prepared using chemical synthesis techniques. Alternatively, nucleic acid molecules of the present invention can be prepared using molecular biology techniques.

[0394] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0395] The above nucleic acid sequence information can be modified for codon preference according to the final host cell (eg, E. coli) expression system to be used.

[0396] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0397] The modified BoNT / A of the present invention can exist in a single-chain or double-chain form. However, the modified BoNT / A preferably exists in a double-chain form, wherein the L chain is connected to the H chain (or its components, such as H) by a disulfide bond. N domain) connection.

[0398] A single-chain modified BoNT / A having a light chain and a heavy chain can be produced using a method comprising expressing a nucleic acid encoding a modified BoNT / A in an expression host, lysing the host cells to provide a host cell homogenate containing the single-chain modified BoNT / A, and isolating the single-chain modified BoNT / A. The single-chain modified BoNT / A described herein can be proteolytically treated using a method comprising contacting the single-chain modified BoNT / A with a protease (e.g., Lys-C) that hydrolyzes a peptide bond in the activation loop of the modified BoNT / A, thereby converting the single-chain modified BoNT / A into a corresponding double-chain modified BoNT / A (e.g., wherein the light chain and the heavy chain are linked together by a disulfide bond). Preferably, a double-chain modified BoNT / A is obtained by this method.

[0399] The term "available" as used herein also encompasses the term "obtained." In one embodiment, the term "available" means obtained.

[0400] Thus, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A produced from a single-chain BoNT / A, wherein the single-chain BoNT / A comprises or consists of a polypeptide sequence described herein. For example, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A produced from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95% or 99.9%) sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A produced from a polypeptide comprising (even more preferably consisting of) SEQ ID NO: 6. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) by a disulfide bond, which can be obtained by a method comprising contacting the single-chain modified BoNT / A with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) by a disulfide bond, which can be obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A to the corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, wherein the L chain is linked to the H chain by a disulfide bond, which can be obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A to the corresponding two-chain modified BoNT / A.

[0401] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to produce a two-chain clostridial neurotoxin are taught in WO 2014 / 080206, WO 2014 / 079495, and EP2677029A2, which are incorporated herein by reference. Lys-C can cleave one or more lysine residues at the C-terminus of the activation loop. When Lys-C cleaves the activation loop more than once, the skilled artisan will understand that the small peptide of the activation loop of the two-chain modified BoNT / A may not be present compared to the SEQ ID NO shown herein.

[0402] As used herein, the term "one or more" may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "one or more" precedes a list, "one or more" may mean all members of the list. Similarly, as used herein, the term "at least one" may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "at least one" precedes a list, "at least one" may mean all members of the list.

[0403] As used herein, the term "disorder" also encompasses "disease." In one embodiment, the disorder is a disease.

[0404] The modified BoNT / A of the present invention can be formulated in any suitable manner for administration to a subject, such as as part of a pharmaceutical composition. Such pharmaceutical compositions comprise the modified BoNT / A of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0405] Liquid dosage forms are typically prepared using a modified BoNT / A and a pyrogen-free sterile vehicle. The modified BoNT / A can be dissolved or suspended in the vehicle, depending on the vehicle and concentration used. In preparing a solution, the modified BoNT / A can be dissolved in the vehicle, the solution can be made isotonic by the addition of sodium chloride if necessary, and sterilized by filtering through a sterile filter using aseptic technique, and then filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics can be dissolved in the vehicle.

[0406] Dry powders are dissolved or suspended in a suitable vehicle prior to use and can be prepared by filling pre-sterilized components into sterile containers using aseptic technique in a sterile area. Alternatively, the components can be dissolved in a suitable container using aseptic technique in a sterile area. The product is then freeze-dried and the container sealed under sterile conditions.

[0407] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile ingredients are suspended in a sterile vehicle, rather than dissolved, and cannot be sterilized by filtration. These ingredients may be isolated under aseptic conditions, or they may be sterilized after isolation, for example, by gamma irradiation.

[0408] Advantageously, a suspending agent such as polyvinylpyrrolidone may be included in the composition to facilitate uniform distribution of the ingredients.

[0409] In one embodiment, the present invention provides a unit dosage form of a modified botulinum neurotoxin A (BoNT / A) for treating upper facial lines comprising:

[0410] a. greater than 73 units of modified BoNT / A; or

[0411] b. greater than 1754 pg (preferably greater than 1800 pg) of modified BoNT / A; and

[0412] c. optionally, a pharmaceutically acceptable carrier, excipient, adjuvant and / or salt,

[0413] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0414] In one aspect, the present invention provides a unit dosage form of a modified botulinum neurotoxin A (BoNT / A) for treating upper facial lines, the unit dosage form comprising:

[0415] a. Greater than 73 units to 332.8 units of modified BoNT / A, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice 50 ) of a modified BoNT / A; or

[0416] b. greater than 1754 pg (preferably greater than 1800 pg) to 8000 pg of modified BoNT / A; and

[0417] c. optionally, a pharmaceutically acceptable carrier, excipient, adjuvant and / or salt,

[0418] d. wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0419] The unit dosage form may include:

[0420] a. 100 units to 200 units of modified BoNT / A; or

[0421] b. 4000 pg to 6000 pg of modified BoNT / A.

[0422] A unit dosage form for treating facial lines can include 73 units to 332.8 units of a modified BoNT / A. The upper limit of the range can be 300, 250, 200, 150, or 100 units of a modified BoNT / A, preferably the upper limit is 200 units. The lower limit of the range can be 80, 90, 100, 150, 200, or 250 units of a modified BoNT / A, preferably the lower limit is 100 units. Preferably, the unit dosage form includes 90 to 250 units of a modified BoNT / A, such as 100 units to 200 units.

[0423] A unit dosage form for treating facial lines can include 1755 pg to 8000 pg of a modified BoNT / A. The upper limit of the range can be 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, or 3000 pg of a modified BoNT / A, with a preferred upper limit of 7000 pg. The lower limit of the range can be 1800, 2000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 pg of a modified BoNT / A, with a preferred lower limit of 2000 pg. Preferably, the unit dosage form includes 2000 pg to 7000 pg of a modified BoNT / A, such as 4,000 pg to 6,000 pg.

[0424] The unit dosage form may contain 1800-7000 pg, 2000-6500 pg, 2500-6000 pg, preferably 4000-5500 pg of modified BoNT / A.

[0425] The unit dosage form may comprise greater than 1754 pg to 8000 pg of a modified BoNT / A. The upper limit of the unit dosage form may be 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 pg of a modified BoNT / A, preferably the upper limit is 5,000 pg. The lower limit of the unit dosage form may be 1,800, 2,000, 2,500, 3000, or 4,000 pg of a modified BoNT / A, preferably the lower limit is 1,800 pg of a modified BoNT / A. The unit dosage form may comprise: 1,800 to 8,000 pg, or 2,000 to 6,000 pg of a modified BoNT / A, more preferably 3,000 to 6,000 pg of a modified BoNT / A. The unit dosage form can be 4,500 to 5,500 pg of modified BoNT / A, such as 4,900 to 5,100 pg of modified BoNT / A.

[0426] The unit dosage form may contain greater than 1800 pg of a modified BoNT / A. The unit dosage form may contain greater than 1800 pg up to 7000 pg of a modified BoNT / A, such as greater than 1800 pg up to 6000 pg of a modified BoNT / A. The unit dosage form may contain greater than 2000 pg up to 5000 pg of a modified BoNT / A.

[0427] The unit dosage form can contain greater than 2555 pg. For example, the unit dosage form can contain greater than 2555 pg up to 8000 pg of modified BoNT / A, such as greater than 2555 pg up to 6000 pg of modified BoNT / A.

[0428] A unit dosage form can contain 2000 pg, 2500 pg, 3000 pg, 4000 pg, 4500 pg, 5000 pg, 5500 pg, 6000 pg, 6500 pg, 7000 pg, 7500 pg, or 8000 pg of a modified BoNT / A.

[0429] More preferably, the unit dosage form can include 2000 to 3500 pg of the modified BoNT / A. For example, the unit dosage form can include 2250 pg to 3250 pg of the modified BoNT / A. Particularly preferred unit dosage forms can include about 2500 pg (e.g., 2500 pg ± 10%) of the modified BoNT / A, for example, the unit dosage form can preferably include 2500 pg of the modified BoNT / A. Another suitable unit dosage form can include about 3000 pg (e.g., 3000 pg ± 10%) of the modified BoNT / A, for example, the unit dosage form can include 3000 pg of the modified BoNT / A.

[0430] The unit dosage form can include 3500-5500 pg of a modified BoNT / A. For example, a unit dosage form can include 3800 pg to 4200 pg of a modified BoNT / A. A unit dosage form can include 4800 to 5200 pg of a modified BoNT / A. A particularly suitable unit dosage form can include about 4000 pg (e.g., 4000 pg ± 10%) of a modified BoNT / A, for example, a unit dosage form can include 4000 pg of a modified BoNT / A. Another particularly preferred unit dosage form can include about 5000 pg (e.g., 5000 pg ± 10%) of a modified BoNT / A, for example, a unit dosage form can preferably include 5000 pg of a modified BoNT / A.

[0431] Also provided is a kit comprising:

[0432] a. a unit dosage form as defined above; and

[0433] b. Instructions for use in the treatment of facial lines; and

[0434] c. Optionally, a diluent.

[0435] Also provided is a kit comprising:

[0436] a. a unit dosage form as defined above; and

[0437] b. Instructions for use for the treatment of frown lines; and

[0438] c. Optionally, a diluent.

[0439] Also provided is a kit comprising:

[0440] a. a unit dosage form as defined above; and

[0441] b. Instructions for use for the treatment of forehead lines; and

[0442] c. Optionally, a diluent.

[0443] Also provided is a kit comprising:

[0444] a. a unit dosage form as defined above; and

[0445] b. Instructions for use for the treatment of lateral canthal lines; and

[0446] c. Optionally, a diluent.

[0447] Also provided is a kit comprising:

[0448] a. a unit dosage form as defined above; and

[0449] b. Instructions for use for the treatment of glabellar lines, forehead lines, and lateral canthal lines; and

[0450] c. Optionally, a diluent.

[0451] The modified BoNT / A in the unit dosage form can comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to any one of SEQ ID NOs: 3-7.

[0452] Preferably, the modified BoNT / A in the unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A can comprise (more preferably consist of) SEQ ID NO: 6.

[0453] Embodiments relating to the various therapeutic uses of the invention are applicable to the methods, compositions (eg, unit dosage forms), and kits of the invention, and vice versa.

[0454] Sequence homology

[0455] Any of a variety of sequence alignment methods can be used to determine percent identity, including but not limited to global methods, local methods, and hybrid methods, such as fragment methods. Protocols for determining percent identity are routine procedures within the capabilities of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding the scores for each residue pair and applying a gap penalty. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, for example, Osamu Gotoh, Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice 264 (4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences.Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8 (5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262 (5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20 (9) Bioinformatics: 1428-1435 (2004).

[0456] Therefore, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In brief, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra), as shown below (amino acids are represented by standard single letter codes).

[0457] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of percent sequence identity can also take into account the number of spaces and the length of each space that needs to be introduced to optimize the comparison of two or more sequences. The determination of percent identity between sequence comparisons and two or more sequences can be performed using specific mathematical algorithms familiar to those skilled in the art (e.g., BLAST).

[0458] Alignment scores used to determine sequence identity

[0459]

[0460] The percent identity was then calculated as follows:

[0461] Total number of identical matches

[0462] ________________________________________________________x100

[0463] [The length of the longer sequence plus

[0464] Number of gaps introduced into longer sequences

[0465] in order to align the two sequences]

[0466] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably slight, i.e. conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity; small deletions, typically 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0467] Conservative amino acid substitution

[0468]

[0469] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace polypeptide amino acid residues. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0470] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methyleneproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, isothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods of incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which a chemically aminoacylated suppressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Non-natural amino acids are incorporated into polypeptides to replace their natural counterparts. See Koide et al., Biochem. 33: 7470-6, 1994. Natural amino acid residues can be converted to non-natural species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitution (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

[0471] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids and unnatural amino acids can replace amino acid residues in the polypeptides of the invention.

[0472] Essential amino acids in the polypeptides of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Biological interaction sites can also be determined by structural physical analysis, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations of putative contact site amino acids. See, for example, de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identity of essential amino acids can also be inferred by homology analysis with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0473] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclosed methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of substitutions allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0474] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 20th Edition, John Wiley and Sons, New York (1994), and Hale & Marham, HarperCollins Dictionary of Biology, Harper Perennial, NY (1991) provide a general dictionary for those of skill in the art for many of the terms used in the present disclosure.

[0475] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to the methods and materials described herein can be used to practice or test the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction.

[0476] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0477] The name of amino acid, three-letter abbreviation or single-letter abbreviation is used herein to refer to amino acid. The term "protein" used herein includes protein, polypeptide and peptide. The term "amino acid sequence" used herein is synonymous with the term "polypeptide" and / or the term "protein". In some cases, "amino acid sequence" is synonymous with the term "peptide". In some cases, "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes of amino acid residues can be used. The three-letter code of amino acid conforms to the definition of the Joint Committee on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0478] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and therefore may vary. It should also be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting, as the scope of the present disclosure is limited only by the attached claims.

[0479] When a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range is also explicitly disclosed (unless the context clearly states otherwise), accurate to one-tenth of the lower limit unit. Each smaller range between any specified value or intermediate value in the specified range and any other specified value or intermediate value in the specified range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the range or excluded from the range, and each range that includes any limit, does not include a limit, or includes two limits in the smaller range is also included in the present disclosure, but does not include any explicitly excluded limits in the range. When the range includes one or two limits, the range excluding one or both of the limits is also included in the present disclosure.

[0480] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a botulinum neurotoxin A" includes a plurality of such candidate drugs and reference to "the botulinum neurotoxin A" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0481] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art for the appended claims.

[0482] BRIEF DESCRIPTION OF THE DRAWINGS

[0483] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0484] Figure 1 FDA-approved treatment for facial wrinkles in adults dose.

[0485] Figure 2 Shown is an SDS-PAGE of purified recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3-4, respectively). Lanes are labeled "Maker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample).

[0486] Figure 3 Cleavage of SNAP-25 in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5 converted to double-chain form, respectively) is shown. Cultured rat primary spinal cord neurons (SCN) were exposed to different concentrations of recombinant BoNT / AB chimeras 1, 2, or 3A for 24 hours at 37°C in a humidified atmosphere of 10% CO2. The cells were then lysed with 1xNuPAGE buffer supplemented with DTT and nuclease (Benzonase). The samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes on a heating block, and then stored at -20°C before analysis of SNAP-25 cleavage by Western blotting. SNAP-25 was detected using a polyclonal antibody that detects both the full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as a secondary antibody.

[0487] Figure 4The mouse toe abduction score test is shown. Under brief general anesthesia, the drug was injected into the gastrocnemius-soleus complex muscle of one hind limb of the mouse; muscle weakness was measured using the toe abduction score (DAS) on a scale of 0-4. The maximum DAS value for each dose was determined and a dose graph was plotted, and the data were fitted to a 4-parameter logistic equation to determine the ED50 and the dose that resulted in DAS4 (DAS 4 dose) values.

[0488] Figure 5 SDS-PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 6 and 7, respectively) is shown. Lanes are labeled "Maker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample).

[0489] Figure 6 Cleavage of SNAP-25 by unmodified BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 2, 6, and 7, respectively) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U-Axiogenesis, Germany) is shown. PERI.4U cells were exposed to different concentrations of recombinant BoNT / A or BoNT / AB chimeras 3B or 3C for 24 hours at 37°C in a humidified CO2 atmosphere with 5% CO2. Cells were then lysed with 1x NuPAGE buffer supplemented with DTT and nuclease (Benzonase). Samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes on a heating block, and stored at -20°C before analysis of SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as a secondary antibody.

[0490] Figure 7 The change of muscle weakness in the digital abduction score test in mice over time is shown. The drug was injected into the gastrocnemius-soleus complex muscle of one hind limb of mice under brief general anesthesia; muscle weakness was measured using the digital abduction score (DAS) on a scale of 0-4. Animals in the lowest dose group that induced a DAS of 4 in the first four days of injection were monitored until muscle weakness fully recovered to a DAS of 0 (no muscle weakness was observed).

[0491] Figure 8 The injection site for modified BoNT / A intramuscular administration for the treatment of glabellar lines is shown.

[0492] Fig. 9Injection sites for modified BoNT / A intramuscular administration for the treatment of glabellar and forehead lines are shown.

[0493] Fig.10 The injection site for modified BoNT / A intramuscular administration when treating only the lateral canthal lines is shown.

[0494] Fig.11 Injection sites for modified BoNT / A intramuscular administration for the treatment of glabellar lines, forehead lines, and lateral canthal lines are shown.

[0495] Sequence Listing

[0496] When a starting Met amino acid residue or a corresponding start codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional.

[0497] SEQ ID NO: 1 (nucleotide sequence of unmodified BoNT / A)

[0498]

[0499]

[0500] SEQ ID NO: 2 (unmodified BoNT / A polypeptide sequence)

[0501]

[0502]

[0503] SEQ ID NO: 3 (Polypeptide sequence of modified BoNT / A "Chimera 1")

[0504]

[0505] SEQ ID NO:4 (Polypeptide sequence of modified BoNT / A "Chimera 2")

[0506]

[0507]

[0508] SEQ ID NO:5 (Polypeptide sequence of modified BoNT / A "Chimera 3A")

[0509]

[0510] SEQ ID NO:6 (Polypeptide sequence of modified BoNT / A "Chimera 3B")

[0511]

[0512]

[0513] SEQ ID NO:7 (Polypeptide sequence of modified BoNT / A "Chimera 3C")

[0514]

[0515] SEQ ID NO: 8 (BoNT / B polypeptide sequence)

[0516]

[0517]

[0518] SEQ ID NO:9—C-terminal L chain fragment

[0519]

[0520] SEQ ID NO:10—C-terminal L chain fragment 2

[0521]

[0522] SEQ ID NO:11—Double-stranded L chain 1

[0523]

[0524] SEQ ID NO:12—Double-stranded L chain 2

[0525]

[0526] SEQ ID NO:13—Double-stranded H chain

[0527] Example

[0528] Example 1

[0529] Cloning, expression and purification of BoNT / AB chimeras

[0530] BoNT / AB chimeric constructs 1, 2, 3A, 3B, and 3C (SEQ ID NOs: 3-7, respectively) were constructed from DNA encoding the parental serotype molecules and appropriate oligonucleotides using standard molecular biology techniques. They were then cloned into 10 The pJ401 expression vector with the A-tag was transformed into BLR(DE3) E. coli cells for overexpression. These cells were cultured in 2L baffled Erlenmeyer flasks at 37°C and 225RPM shaking, containing 1L of modified TerrificBroth (mTB) supplemented with appropriate antibiotics. Once A 600 Once the pH reached >0.5, the incubator temperature was lowered to 16 °C and then induced one hour later with 1 mM IPTG for 20 h with shaking at 225 RPM to express the recombinant BoNT / AB construct.

[0531] The harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimeric molecules were then extracted in ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques. This involved capture using a hydrophobic interaction resin and an intermediate purification step using an anion exchange resin. The partially purified molecules were then proteolytically cleaved with endoproteinase Lys-C to generate an active duplex. Further purification was performed with a second hydrophobic interaction resin to obtain the final BoNT / AB chimera.

[0532] For the case with a ten-histidine tag (H 10 ) (Chimera 1, 2, 3A) of the BoNT / AB chimeric molecule, the capture step uses immobilized nickel resin instead of hydrophobic interaction resin. .

[0533] The sequence of each chimera is shown in Table 1.

[0534]

[0535] Table 1 - Chimeric BoNT / AB Constructs

[0536] Example 2

[0537] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0538] With C-terminal His 10 The tags and E1191M / S1199Y double mutant BoNT / AB chimeras 1, 2 and 3A were prepared according to Example 1 ( Figure 2 ) and tested for functional activity.

[0539] SNAP-25 cleavage assay in rat spinal cord neurons

[0540] Primary cultures of rat spinal cord neurons (SCN) were prepared and cultured in 96-well tissue culture plates for 3 weeks (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B; and Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). Serial dilutions of BoNT / AB were prepared in SCN feed medium. Growth medium from wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. 125 μL of diluted toxin was then added to the plate (triplicate wells). Treated cells were incubated at 37°C, 10% CO2 for 24 ± 1 h).

[0541] Analysis of BoNT activity using SNAP-25 cleavage assay

[0542] After treatment, BoNT was removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was probed with a SNAP-25-specific primary antibody (Sigma # S9684), which recognizes uncut SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma # A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK), and the percentage of SNAP-25 cut at each BoNT concentration was calculated. Data were fitted to a 4-parameter logistic equation, and pEC was calculated using GraphPad Prism version 6 (GraphPad). 50。

[0543] Table 2 below provides the pEC values ​​of chimeras 1, 2, and 3A determined in the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retain the ability to enter rat spinal cord neurons and cleave their target substrates. However, chimera 3A was more potent than chimeras 1 and 2 in this assay (see also Figure 3 ).

[0544] <![CDATA[pEC 50 ±SEM]]> Chimera 1 12.42±0.04 Chimera 2 12.57±0.01 Chimera 3A 12.89±0.04

[0545] Table 2. pEC 50 value.

[0546] Digital abduction score (DAS) measurement

[0547] The method for measuring the activity of BoNT / AB chimeras 1, 2 and 3A in the DAS assay is based on the startle toe extension reflex of mice when they are briefly suspended by their tails. This reflex, recorded as the digital abduction score (DAS), is inhibited after BoNT is injected into the gastrocnemius-soleus muscles of the hind paw. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animals extend their hind limbs and abduct their hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6(Supplement 4) S3-S10).

[0548] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an anesthesia chamber. Each mouse was injected intramuscularly in the gastrocnemius-soleus muscle of the right hind paw with BoNT / AB chimeras or vehicle (phosphate buffered saline containing 0.2% gelatin).

[0549] After injection of the neurotoxin, different degrees of toe abduction were scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in toe abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the mean of the maximal effects of each dose. The mathematical model used was a 4-parameter logistic model.

[0550] DAS was performed every 2 hours on the first day after taking the drug; thereafter, it was performed 3 times a day for 4 days.

[0551] Figure 4 The fitted curves for chimeras 1, 2 and 3A (SEQ ID NOs: 3, 4 and 5 converted to double-stranded form, respectively) are shown. The chimera 3A curve is shifted to the left, which means that a lower dose of chimera 3A achieves a similar DAS response compared to chimeras 1 and 2, thus indicating that chimera 3A is more effective than the other chimeras in the mouse DAS assay; see also the table below (Table 3), which provides the calculated ED50 values ​​for each chimera and the dose that resulted in DAS 4 (the highest score).

[0552] Table 3 below provides the ED values ​​determined in the mouse DAS assay for unmodified recombinant BoNT / A1 (rBoNT / A1 - SEQ ID NO: 2 converted to a double-chain form) and Chimeras 1, 2, and 3A. 50 and DAS 4 doses. These results indicate that among the three chimeras, chimera 3A has the highest potency in inducing muscle weakness in vivo. Figure 4 The studies shown in and Table 3 were performed in mice obtained from Charles River Laboratories.

[0553]

[0554] Table 3. ED 50 value.

[0555] Example 3

[0556] Comparison of BoNT / AB chimeras 3B and 3C with unmodified BoNT / A1

[0557] Unlabeled BoNT / AB chimeras 3B and 3C (with and without the E1191M / S1199Y double mutation, respectively) (SEQ ID NOs: 6 and 7) were prepared as described in Example 1 ( Figure 5 ) and tested for functional activity using unmodified BoNT / A (SEQ ID NO: 2 converted into a double-chain form) as a reference. Human pluripotent stem cell SNAP-25 cleavage assay

[0558] Cryopreserved PERI.4U cells were purchased from Axiogenesis (Cologne, Germany). Cell thawing and plating were performed according to the manufacturer's recommendations. Briefly, cryovials containing cells were thawed in a 37°C water bath for 2 min. After gentle resuspension, cells were transferred to a 50 mL tube. 1 mL of Thaw medium to wash the cryovials, then transfer the medium dropwise to the cell suspension in the 50 mL tube, and then transfer 2 mL Thaw medium was added dropwise to the 50 mL tube. The cells were then counted using a hemocytometer. Afterwards, another 6 mL Thaw medium is added to the cell suspension. Centrifuge at 260 x g (e.g., 1,100 RPM) for 6 minutes at room temperature to obtain a cell pellet. Then resuspend the cells in the complete The cells were cultured at 50,000 to 150,000 cells / cm 2 The cells were seeded at a density of 1.5 ng / mL on cell culture plates coated with poly-L-ornithine and laminin. The cells were cultured at 37°C in a humidified CO2 atmosphere, and the culture medium was completely replaced every 2-3 days during the culture period.

[0559] For toxin treatment, Prepare serial dilutions of BoNT in culture medium. Collect and filter the culture medium in the wells to be treated (0.2 μm filter). Add 125 μL of filtered culture medium back to each test well. Then add 125 μL of diluted toxin to the plate (triplicate wells). Incubate treated cells at 37°C, 10% CO2 for 48 ± 1 h).

[0560] Analysis of BoNT activity using SNAP-25 cleavage assay

[0561] After treatment, BoNT was removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1xNuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL nuclease (benzonase) (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was probed with a SNAP-25-specific primary antibody (Sigma # S9684), which recognizes uncut SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma # A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK), and the percentage of SNAP-25 cut at each BoNT concentration was calculated. Data were fitted to a 4-parameter logistic equation, and pEC was calculated using GraphPad Prism version 6 (GraphPad). 50 .

[0562] Figure 6 The chimeras 3B and 3C showed a stronger potency than rBoNT / A1 in cleaving SNAP-25 in induced human pluripotent stem cells, but the former was significantly more potent. This could be explained by the double mutation, which increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells ( Figure 6 , Table 4).

[0563] <![CDATA[pEC 50 ±SEM]]> rBoNT / A1 10.21±0.05 Chimera 3B 12.38±0.06 Chimera 3C 10.72±0.08

[0564] Table 4. pEC 50 value.

[0565] Digital Abduction Score (DAS) Test – Safety Ratio

[0566] The method for measuring BoNT activity in the DAS assay is based on the startle toe extension reflex of mice when they are briefly suspended by their tails. This reflex is recorded as the digital abduction score (DAS) and is inhibited after BoNT is injected into the gastrocnemius-soleus muscles of the hind paw. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animals extend their hind limbs and abduct their hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6(Suppl 4) S3-S10).

[0567] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an anesthesia chamber. Each mouse was injected intramuscularly with BoNT or vehicle (phosphate buffered saline containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind paw.

[0568] After injection of the neurotoxin, different degrees of toe abduction were scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in toe abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the mean of the maximal effects of each dose. The mathematical model used was a 4-parameter logistic model.

[0569] DAS was performed every 2 hours on the first day after dosing; thereafter, DAS was performed 3 times a day for 4 days for all doses. Animals injected with vehicle and the lowest dose group that induced a DAS of 4 four days before injection were subsequently monitored until muscle weakness fully recovered to a DAS of 0 (no muscle weakness was observed).

[0570] To calculate the safety ratio, all animals were weighed one day before toxin injection (D0) and thereafter once daily throughout the study. The mean body weight, standard deviation, and standard error mean were calculated for each dose group daily. To obtain the safety ratio (-10% ΔBW / ED 50 ), the dose at which the mean body weight of a dose group at any time during the study was less than 10% of the mean body weight of the same dose group at D0 divided by the ED value of the BoNT being studied 50 The lethal dose was defined as the dose at which one or more animals in the dose group died.

[0571] Figure 7 The duration of muscle weakness in a mouse digital abduction scoring assay is shown over time for unmodified BoNT / A, chimera 3B, and chimera 3C (SEQ ID NOs: 2, 6, and 7 converted to double-chain form), indicating a longer duration of effect for the chimeras.

[0572] Table 5 below provides the ED values ​​for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50and DAS 4 doses. The table also provides the total duration of action from DAS 4 dose until myasthenia is completely restored to DAS 0 (no myasthenia is observed). In addition, the table also shows the lethal dose and safety ratio (-10% ΔBW / ED 50 ), as defined above. Compared with rBoNT / A1, chimeras 3B and 3C had a longer duration of action, a better safety profile, and a higher lethal dose. Figure 7 The studies shown in and Table 5 were performed in mice obtained from the Janvier laboratory.

[0573]

[0574] Table 5. DAS and safety ratios of BoNT / AB chimeras.

[0575] Example 4

[0576] Dosage regimen for the treatment of glabellar lines

[0577] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials, each containing 15 ng of modified BoNT / A. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0578] Moderate to severe frown lines Figure 8 Treatment was performed with the indicated injection regimen.

[0579] The unit dose is 20-300 pg (2-35 units).

[0580] Intramuscular injections may be given at up to 5 sites per unit dose, with a maximum total dose of 1500 pg (177 units).

[0581] Example 5

[0582] Dosage regimen for the treatment of glabellar and forehead lines

[0583] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials, each containing 15 ng of modified BoNT / A. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0584] Moderate to severe frown lines and forehead lines Fig. 9 Treatment was performed with the indicated injection regimen.

[0585] The unit dose is 20-300 pg (2-35 units).

[0586] Intramuscular injections may be given as unit doses at up to 10 sites, with a maximum total dose of 3000 pg (355 units).

[0587] Example 6

[0588] Dosage regimen for the treatment of lateral canthal wrinkles

[0589] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials, each containing 15 ng of modified BoNT / A. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0590] Moderate to severe lateral canthal wrinkles Fig.10 Treatment was performed with the indicated injection regimen.

[0591] The unit dose is 20-300 pg (2-35 units).

[0592] Intramuscular injections may be given at up to 6 sites per unit dose, with a maximum total dose of 1800 pg (213 units).

[0593] Example 7

[0594] Dosage regimen for the treatment of glabellar lines, forehead lines, and lateral canthal lines

[0595] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials, each containing 15 ng of modified BoNT / A. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0596] Moderate to severe frown lines, forehead lines and lateral canthal lines Fig.11 Treatment was performed with the indicated injection regimen.

[0597] The unit dose is 20-300 pg (2-35 units).

[0598] Intramuscular injections may be given at up to 16 sites per unit dose, with a maximum total dose of 4800 pg (569 units).

[0599] Example 8

[0600] Further characterization of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form)

[0601] Modified BoNT / A (e.g., BoNT / AB chimera) converted to a two-chain form SEQ ID NO: 6 in mouse LD 50 The assay was tested and the result was 1.202 ng / kg. Therefore, 1 unit of SEQ ID NO: 6 corresponds to 24.04 pg in this assay.

[0602] In addition, the BoNT / AB chimeras were tested in the rat DAS assay to determine The duration of action compared (according to Example 6). The results are listed in the following Table 6:

[0603]

[0604] Table 6. Duration of action.

[0605] In conclusion, the duration of action of BoNT / AB is is much longer and similar to SEQ ID NO: 6. Therefore, it is expected that the unit dose and dosing regimen of SEQ ID NO: 6 can be similarly applied to BoNT / AB to provide better facial wrinkle treatment.

[0606] Example 9

[0607] Calculation of unit dose of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form) for upper facial lines

[0608] Based on preclinical pharmacology data, a unit dose range (UD) of the modified BoNT / A suitable for human use was calculated.

[0609] The DAS ED of SEQ ID NO:6 was calculated. 50 13pg / kg. 50 The lowest pharmacologically active dose is considered to be approximately 300 times lower than the no observed adverse effect level (NOAEL) of 4 ng / kg in the same animal species. 50 It is 13pg / kg, which is equivalent to a dose of 0.8ng for a person weighing 60kg.

[0610] Therefore, the lower limit of the unit dose was selected as 20 pg and the upper limit was 1500 pg, which is lower than the NOAEL 4 ng / kg in two non-clinical safety species (rats and monkeys) converted to a human dose of 60 kg body weight.

[0611] Given the improved safety profile, the maximum total dose for the treatment of upper facial wrinkles was set at 24,000 pg, which was calculated based on the NOAEL of 4 ng / kg in non-clinical safety species (rats and monkeys) and converted to a human dose of 60 kg body weight.

[0612] Example 10

[0613] Dosing regimen for treating glabellar lines using modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form)

[0614] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0615] Moderate to severe frown lines Figure 8 Treatment was performed with the indicated injection regimen.

[0616] The unit dose is 20-1500 pg (0.8-62 units).

[0617] Intramuscular injections were given at a maximum of 5 sites per unit dose, with a maximum total dose of 7500 pg (312 units).

[0618] Embodiment 11

[0619] Administration of modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form) for the treatment of glabellar and forehead lines plan

[0620] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0621] Moderate to severe frown lines and forehead lines Fig. 9 Treatment was performed with the indicated injection regimen.

[0622] The unit dose is 20-1500 pg (0.8-62 units).

[0623] Intramuscular injections may be given as unit doses at up to 10 sites, with a maximum total dose of 15,000 pg (624 units).

[0624] Example 12

[0625] Dosing regimen for treating lateral canthal wrinkles using modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form)

[0626] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0627] Moderate to severe lateral canthal wrinkles Fig.10 Treatment was performed with the indicated injection regimen.

[0628] The unit dose is 20-1500 pg (0.8-62 units).

[0629] Intramuscular injections may be given at up to 6 sites per unit dose, with a maximum total dose of 9000 pg (374 units).

[0630] Example 13

[0631] Treatment of glabellar lines, forehead lines and lateral canthus using modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form) Dosage regimen for

[0632] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0633] Moderate to severe frown lines, forehead lines and lateral canthal lines Fig.11 Treatment was performed with the indicated injection regimen.

[0634] The unit dose is 20-1500 pg (0.8-62 units).

[0635] Intramuscular injections may be given at up to 16 sites per unit dose, for a maximum total dose of 24,000 pg (998 units).

[0636] Embodiment 14

[0637] Safety and efficacy of modified BoNT / A (converted to a double-chain form of SEQ ID NO: 6) in humans

[0638] A comprehensive Phase I / II multicenter, double-blind, randomized, Dysport- and placebo-controlled, dose-escalation and dose-finding study was conducted to evaluate the safety and efficacy of modified BoNT / A (SEQ ID NO:6 converted to di-chain form) in the treatment of moderate to severe upper facial wrinkles in adults. The upper facial wrinkles included glabellar lines, forehead lines, and lateral canthal lines. The study included human sequential dose escalation in a unique patient cohort with adult upper facial wrinkles. The upper facial wrinkles included glabellar lines, forehead lines, and lateral canthal lines. A unit dose of modified BoNT / A, Dysport, or placebo was injected intramuscularly per muscle. The 6 cohorts were administered different (increasing) amounts of modified BoNT / A (SEQ ID NO:6 converted to di-chain form). The total dose ranged from 0.1 to 6.0 ng. The modified BoNT / A was injected into up to 16 sites on the upper face (5 sites on glabellar lines, 5 sites on forehead lines, and 3 sites on each side of the lateral canthal lines - see Fig.11 ).

[0639] The results show that despite the extremely high dose per muscle, all modified BoNT / A unit doses and total doses tested (i.e., up to 6,000 pg) were effective, safely tolerated, and no adverse reactions were observed. This indicates that the modified BoNT / A does not spread from the injection site and highlights the excellent safety profile of modified BoNT / A (SEQ ID NO: 6). Based on these findings, it is believed that higher unit doses can be administered per muscle without adverse reactions. In addition, given that the toxin does not spread systemically, it is believed that up to 13-14 times the unit dose can be administered without safety issues.

[0640] Therefore, we choose a unit dose of up to 5,000 pg + / - 10%, with a maximum of 16 injections in the upper facial area (e.g., five sites on the glabellar lines, five sites on the forehead lines, and three sites on both sides of the lateral canthal lines) for the treatment of upper facial lines. Therefore, the total dose injected during the treatment period will be up to 80,000 (+ / - 10%) respectively.

[0641] For example, unit doses up to 5,000 pg + / - 10% have been selected to treat glabellar lines (two for each corrugator supercilii and one for the procerus), with a total dose to the glabellar region of up to 25,000 pg, which is approximately 10-fold lower than the NOAEL of 4 ng / kg in two nonclinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

[0642] Indeed, as explained in Example 21, five injections (two in each corrugator supercilii and one in the procerus) of a 4,000 pg unit dose each (to provide a total dose of 20,000 pg) were well tolerated.

[0643] Embodiment 15

[0644] Dosing regimen for treating glabellar lines using modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form)

[0645] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0646] Moderate to severe frown lines Fig.11 Treatment was performed according to the indicated injection schedule. That is, a maximum of two unit doses were injected into each corrugator muscle and one unit dose was injected into the procerus muscle.

[0647] The unit dose ranged from >1754 pg to 5,000 ng. The total dose of modified BoNT / A administered during treatment was up to 88,000 pg. This is The maximum total dose that can be administered when treating facial lines is more than 14 times higher without approaching the toxicity limit (a concern with traditional treatment regimens). Therefore, clinicians are able to tailor treatment regimens to their patients, knowing that a total dose of 88,000 pg can be administered without concern for toxicity, allowing for the treatment of additional muscles in the subject and / or ensuring that each muscle and / or site receives an effective dose.

[0648] Example 16

[0649] Administration of modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form) for the treatment of glabellar and forehead lines plan

[0650] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0651] Moderate to severe frown lines and forehead lines Fig. 9 Treatment was performed according to the indicated injection schedule. That is, a maximum of two unit doses were injected into each corrugator muscle, one unit dose was injected into the procerus muscle, and a maximum of five unit doses were injected into the frontalis muscle.

[0652] The unit dose ranged from >1754 pg to 5,000 ng. The total dose of modified BoNT / A administered during treatment was up to 88,000 pg. This is The maximum total dose that can be administered when treating facial wrinkles is more than 14 times higher without approaching the toxicity limit (a concern with traditional treatment regimens). Therefore, clinicians are able to tailor treatment regimens to their patients, knowing that a total dose of 88,000 pg can be administered without concern for toxicity, allowing for the treatment of additional muscles in the subject and / or ensuring that each muscle and / or site receives an effective dose.

[0653] Embodiment 17

[0654] Dosing regimen for treating lateral canthal wrinkles using modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form)

[0655] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0656] Moderate to severe lateral canthal wrinkles Fig.10 Treatment was performed using the indicated injection schedule, i.e., a maximum of three unit doses were injected per orbicularis oculi muscle.

[0657] The unit dose ranged from >1754 pg to 5,000 ng. The total dose of modified BoNT / A administered during treatment was up to 88,000 pg. This is The maximum total dose that can be administered when treating facial wrinkles is more than 14 times higher without approaching the toxicity limit (a concern with traditional treatment regimens). Therefore, clinicians are able to tailor treatment regimens to their patients, knowing that a total dose of 88,000 pg can be administered without concern for toxicity, allowing for the treatment of additional muscles in the subject and / or ensuring that each muscle and / or site receives an effective dose.

[0658] Embodiment 18

[0659] Treatment of glabellar lines, forehead lines and lateral canthus using modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form) Dosage regimen for

[0660] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0661] Moderate to severe frown lines, forehead lines and lateral canthal lines Fig.11 The treatment was carried out according to the injection schedule shown. That is, a maximum of two unit doses were injected into each corrugator muscle, one unit dose was injected into the procerus muscle, a maximum of five unit doses were injected into the frontalis muscle, and a maximum of three unit doses were injected into each orbicularis oculi muscle.

[0662] The unit dose ranged from >1754 pg to 5,000 ng. The total dose of modified BoNT / A administered during treatment was up to 88,000 pg. This is The maximum total dose that can be administered when treating facial wrinkles is more than 14 times higher without approaching the toxicity limit (a concern with traditional treatment regimens). Therefore, clinicians are able to tailor treatment regimens to their patients, knowing that a total dose of 88,000 pg can be administered without concern for toxicity, allowing for the treatment of additional muscles in the subject and / or ensuring that each muscle and / or site receives an effective dose.

[0663] Embodiment 19

[0664] Treatment of patients with upper facial lines

[0665] John, 55 years old, suffers from severe glabellar lines, forehead lines, and lateral canthal lines. Modified BoNT / A (SEQ ID NO: 6) was administered in one or more unit doses (1 x unit dose = 5000 pg) to the following muscles:

[0666] - Two unit doses (therefore four unit doses on the corrugator supercilii muscle) to one site on each of the corrugator supercilii muscle and procerus muscle for the treatment of glabellar lines,

[0667] - five unit doses to the frontalis muscle for the treatment of forehead lines; and

[0668] - Three unit doses (thus six unit doses injected on the orbicularis oculi muscle) to the outside of each orbicularis oculi muscle for the treatment of lateral canthal lines.

[0669] The total dose administered was 80,000 ng (+ / - 10%) modified BoNT / A (SEQ ID NO: 6 converted to double-chain form).

[0670] All lines (wrinkles) were alleviated, the skin looked clear and youthful, and due to the long duration of the modified BoNT / A, John did not require further treatment for more than 9 months. As a result, John received less frequent injections (e.g., annually) than the same subjects treated with unmodified BoNT / A. In addition, Bill did not show any side effects due to the improved safety profile of the modified BoNT / A.

[0671] Embodiment 20

[0672] Safety and efficacy of modified BoNT / A (converted to a double-chain form of SEQ ID NO: 6) in humans SEQ ID NO: 6 (converted to a double-stranded form) was administered to human subjects by intramuscular injection. The subjects were administered 2 x 15,000 pg unit doses (i.e., 30,000 pg total), 2 x 25,000 pg unit doses (i.e., 50,000 pg total), or 2 x 36,000 pg unit doses (i.e., 72,000 pg total) of the modified BoNT / A (SEQ ID NO: 6 converted to a double-stranded form).

[0673] The results showed that all modified BoNT / A unit doses tested were effective in paralyzing muscles, safely tolerated, and no adverse reactions were observed, despite extremely high doses per muscle (e.g., 25,000 pg and 36,000 pg unit doses). This indicates that the modified BoNT / A does not diffuse from the injection site and highlights the excellent safety of the modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form).

[0674] The unit doses and total doses described herein in the context of treating facial wrinkles are well within the dosage range that is safe and effective for such advantageous modified BoNT / A molecules described herein, providing clinicians with flexibility in treatment options, including taking advantage of the advantageous properties and excellent safety profile of modified BoNT / As (e.g., SEQ ID NO: 6 converted to a two-chain form).

[0675] Embodiment 21

[0676] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form) in the treatment of glabellar lines Effectiveness

[0677] SEQ ID NO: 6 (converted to double-chain form) was administered to human subjects by intramuscular injection.

[0678] One group of subjects received five injections (two injections into each corrugator supercilii muscle and one injection into each procerus muscle) at a unit dose of 2,500 pg per injection for a total dose of 12,500 pg, while the other group received five injections (two injections into each corrugator supercilii muscle and one injection into each procerus muscle) at a unit dose of 4,000 pg per injection for a total dose of 20,000 pg.

[0679] Glabellar lines were reduced. The results showed that all modified BoNT / A unit doses tested were safely tolerated and no adverse effects were observed, despite the very high dose per muscle. This suggests that the modified BoNT / A does not diffuse from the injection site and highlights the excellent safety profile of the modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form).

[0680] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to these specific embodiments. In fact, various modifications of the modes of implementing the present invention will be apparent to those skilled in the art of biochemistry and biotechnology or related fields, and these modifications are intended to fall within the scope of the following claims.

Claims

1. A modified BoNT / A for use in treating facial wrinkles, wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject, wherein the modified BoNT / A is administered at a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of the modified BoNT / A per site, in, Multiple sites are selected from: Up to two sites on the corrugator supercilii muscle and up to one site on the procerus supercilii muscle for the treatment of frown lines, Up to five sites on the frontalis muscle for forehead lines; and Up to three sites on the orbicularis oculi muscle for the treatment of lateral canthal lines, wherein the total dose of modified BoNT / A administered during the treatment period is up to 88,000 pg, and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

2. The modified BoNT / A for use according to claim 1, wherein the unit dose of the modified BoNT / A is greater than 1754 pg, and wherein the unit dose of the modified BoNT / A is up to 8,000 pg of modified BoNT / A.

3. The modified BoNT / A for use according to claim 1 or claim 2, wherein the unit dose of the modified BoNT / A is greater than 1754 pg, and wherein the unit dose of the modified BoNT / A is up to 7,500 pg of modified BoNT / A.

4. The modified BoNT / A for use according to any of the preceding claims, wherein the unit dose of the modified BoNT / A is greater than 1754 pg, and wherein the unit dose of the modified BoNT / A is up to 5,000 pg of modified BoNT / A.

5. The modified BoNT / A for use according to any one of the preceding claims, wherein the unit dose is 2000 to 3500 pg of the modified BoNT / A, preferably wherein the unit dose is 2250 to 3250 pg of the modified BoNT / A.

6. The modified BoNT / A for use according to any one of the preceding claims, wherein the unit dose is about 2500 pg of the modified BoNT / A, preferably wherein the unit dose is 2500 pg of the modified BoNT / A.

7. The modified BoNT / A for use according to any one of claims 1 to 4, wherein the unit dose is 3500-5500 pg of the modified BoNT / A, preferably wherein the unit dose is 3800 pg to 4200 pg of the modified BoNT / A.

8. The modified BoNT / A for use according to any one of claims 1 to 4 or 7, wherein the unit dose is about 4000 pg of the modified BoNT / A, preferably wherein the unit dose is 4000 pg of the modified BoNT / A.

9. The modified BoNT / A for use according to any of the preceding claims, wherein the total dose of the modified BoNT / A administered during treatment is up to 80,000 pg.

10. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A has a safety ratio greater than 7, wherein the safety ratio is calculated as: the toxin dose required for -10% body weight change measured in pg / mouse divided by the DAS ED measured in pg / mouse. 50 , where ED 50 = the dose required to produce a DAS score of 2.

11. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A is used to treat glabellar lines, preferably wherein the modified BoNT / A is administered to two sites in the corrugator supercilii muscle and one site in the procerus procerus muscle, more preferably wherein the modified BoNT / A is administered to a total of five sites.

12. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A is used to treat glabellar lines, preferably wherein the modified BoNT / A is administered to two sites in each of the corrugator supercilii muscle and one site in the procerus procerus muscle, more preferably wherein the modified BoNT / A is administered to a total of five sites.

13. The modified BoNT / A for use according to claim 11 or claim 12, wherein the total dose administered to the glabellar lines is 12,000 pg to 13,000 pg of the modified BoNT / A; preferably wherein the unit dose is 2000 pg to 3000 pg of the modified BoNT / A, more preferably wherein the unit dose is 2500 pg of the modified BoNT / A.

14. The modified BoNT / A for use according to any one of claims 11-13, wherein the total dose administered to the glabellar lines is about 12,500 pg of the modified BoNT / A, preferably wherein the total dose administered to the glabellar lines is 12,500 pg of the modified BoNT / A.

15. The modified BoNT / A for use according to claim 11 or claim 12, wherein the total dose administered to the glabellar lines is 19,000 pg to 21,000 pg of the modified BoNT / A; preferably wherein the unit dose is 3500 to 4500 pg of the modified BoNT / A, more preferably wherein the unit dose is 4000 pg of the modified BoNT / A.

16. The modified BoNT / A for use according to any one of claims 11-12 or 15, wherein the total dose administered to the glabellar lines is about 20,000 pg of the modified BoNT / A, preferably wherein the total dose administered to the glabellar lines is 20,000 pg of the modified BoNT / A.

17. The modified BoNT / A for use according to any one of claims 1-10, wherein the modified BoNT / A is used to treat forehead lines, preferably wherein the modified BoNT / A is administered to five sites of the frontalis muscle.

18. The modified BoNT / A for use according to claim 17, wherein the total dose administered to the forehead lines is 12,000 pg to 13,000 pg of the modified BoNT / A; preferably wherein the unit dose is 2000 pg to 3000 pg of the modified BoNT / A, more preferably wherein the unit dose is 2500 pg of the modified BoNT / A.

19. The modified BoNT / A for use according to any one of claims 17-18, wherein the total dose administered to the forehead lines is about 12,500 pg of the modified BoNT / A, preferably wherein the total dose administered to the forehead lines is 12,500 pg of the modified BoNT / A.

20. The modified BoNT / A for use according to claim 17, wherein the total dose administered to the forehead lines is 19,000 pg to 21,000 pg of the modified BoNT / A; preferably wherein the unit dose is 3500 pg to 4500 pg of the modified BoNT / A, more preferably wherein the unit dose is 4000 pg of the modified BoNT / A.

21. The modified BoNT / A for use according to any one of claims 17 or 20, wherein the total dose administered to the forehead lines is about 20,000 pg of the modified BoNT / A, preferably wherein the total dose administered to the forehead lines is 20,000 pg of the modified BoNT / A.

22. The modified BoNT / A for use according to any one of claims 1-10, wherein the modified BoNT / A is used to treat lateral canthal wrinkles, preferably wherein the modified BoNT / A is administered to three sites (e.g., on the outer portion thereof) of the orbicularis oculi muscle to treat lateral canthal wrinkles, more preferably wherein the modified BoNT / A is administered to a total of six sites.

23. The modified BoNT / A for use according to any one of claims 1-10 or 22, wherein the modified BoNT / A is used to treat lateral canthal wrinkles, preferably wherein the modified BoNT / A is administered to three sites per orbicularis oculi muscle (e.g., on the exterior of each orbicularis oculi muscle) to treat lateral canthal wrinkles.

24. The modified BoNT / A for use according to claim 22 or claim 23, wherein the total dose administered to the lateral canthal line is 14,000 pg to 16,000 pg of the modified BoNT / A; preferably wherein the unit dose is 2000 to 3000 pg of the modified BoNT / A, more preferably wherein the unit dose is 2500 pg of the modified BoNT / A.

25. The modified BoNT / A for use according to any one of claims 22 to 24, wherein the total dose administered to the lateral canthal line is about 15,000 pg of the modified BoNT / A, preferably wherein the total dose administered to the lateral canthal line is 15,000 pg of the modified BoNT / A.

26. The modified BoNT / A for use according to any one of claims 22 to 23, wherein the total dose administered to the lateral canthal line is 23,000 pg to 25,000 pg of the modified BoNT / A; preferably wherein the unit dose is 3500 pg to 4500 pg of the modified BoNT / A, more preferably wherein the unit dose is 4000 pg of the modified BoNT / A.

27. The modified BoNT / A for use according to any one of claims 22 to 23, wherein the total dose administered to the lateral canthal line is about 24,000 pg of the modified BoNT / A, preferably wherein the total dose administered to the lateral canthal line is 24,000 pg of the modified BoNT / A.

28. The modified BoNT / A for use according to any one of claims 1 to 10, wherein the modified BoNT / A is used to treat glabellar lines, forehead lines and lateral canthal lines, preferably wherein: - administering the modified BoNT / A to two sites of the corrugator supercilii muscle and one site of the procerus supercilii muscle for treating glabellar lines; - administering the modified BoNT / A to five sites of the frontalis muscle; and - The modified BoNT / A was applied to three sites outside the orbicularis oculi muscle for the treatment of lateral canthal wrinkles.

29. The modified BoNT / A for use according to any one of the preceding claims, wherein the modified BoNT / A is used to treat glabellar lines, forehead lines and lateral canthal lines, preferably wherein: - applying the modified BoNT / A to two sites on each of the corrugator supercilii muscle and one site on the procerus supercilii muscle for treating glabellar lines; - administering the modified BoNT / A to five sites in the frontalis muscle; and - Administering the modified BoNT / A to three sites on each orbicularis oculi muscle (preferably on the outer part of each orbicularis oculi muscle) for the treatment of lateral canthal wrinkles.

30. The modified BoNT / A for use according to claim 28 or claim 29, wherein the total dose for treating glabellar lines, forehead lines, and lateral canthal lines is 43,000 to 44,000 pg of the modified BoNT / A; preferably wherein the unit dose is 2000 to 3000 pg of the modified BoNT / A, more preferably wherein the unit dose is 2500 pg of the modified BoNT / A.

31. The modified BoNT / A for use according to any one of claims 28-30, wherein the total dose for treating glabellar lines, forehead lines, and lateral canthal lines is about 44,000 pg of modified BoNT / A, preferably wherein the total dose for treating glabellar lines, forehead lines, and lateral canthal lines is 44,000 pg of modified BoNT / A.

32. The modified BoNT / A for use according to claim 28 or claim 29, wherein the total dose for treating glabellar lines, forehead lines, and lateral canthal lines is 63,000 to 65,000 pg of the modified BoNT / A; preferably wherein the unit dose is 3500 to 4500 pg of the modified BoNT / A, more preferably wherein the unit dose is 4000 pg of the modified BoNT / A.

33. The modified BoNT / A for use according to claim 28, 29 or 32, wherein the total dose used to treat glabellar lines, forehead lines and lateral canthal lines is about 64,000 pg of modified BoNT / A, preferably wherein the total dose used to treat glabellar lines, forehead lines and lateral canthal lines is 64,000 pg of modified BoNT / A.

34. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A is administered to 8, 10, 12, 14, or 16 sites.

35. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A is administered to 7, 9, 11, 13, or 15 sites.

36. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A comprises a polypeptide sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:6; preferably wherein the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:

6.

37. The modified BoNT / A for use according to any of the preceding claims, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / BH C domain, and wherein 24.04 pg of the modified BoNT / A corresponds to a calculated median lethal dose (LD50) in mice 50 ) (e.g., and wherein 1 Unit is the amount of the modified BoNT / A corresponding to the calculated median lethal dose (LD50) in mice).

38. A unit dosage form of a modified botulinum neurotoxin A (BoNT / A), the unit dosage form comprising: a. Greater than 73 units of modified BoNT / A, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice 50 ) of a modified BoNT / A; or b. greater than 1754 pg (preferably greater than 1800 pg) of modified BoNT / A; and c. optionally, a pharmaceutically acceptable carrier, excipient, adjuvant and / or salt, The modified BoNT / A comprises a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

39. A unit dosage form according to claim 38 comprising: a. greater than 73 units and up to 332.8 units of modified BoNT / A; or b. Greater than 1754 pg (preferably greater than 1800 pg) and up to 8000 pg of modified BoNT / A.

40. The unit dose according to claim 38 or claim 39, wherein the unit dosage form comprises 2000 pg to 3500 pg of modified BoNT / A, preferably 2250 pg to 3250 pg of modified BoNT / A.

41. The unit dosage form according to any one of claims 38 to 39, wherein the unit dosage form comprises about 2500 pg of a modified BoNT / A, preferably wherein the unit dosage form comprises 2500 pg of a modified BoNT / A.

42. The unit dosage form according to claim 38 or claim 39, wherein the unit dosage form comprises 3500 pg to 5500 pg of modified BoNT / A, preferably 3800 pg to 4200 pg of modified BoNT / A.

43. The unit dosage form according to any one of claims 38, 39 or 42, wherein the unit dosage form comprises about 4000 pg of a modified BoNT / A, preferably wherein the unit dosage form comprises 4000 pg of a modified BoNT / A.

44. A kit comprising: a. A unit dosage form according to any one of claims 38-43; as well as b. instructions for use for treating facial lines (e.g., frown lines, forehead lines, and / or lateral canthal lines); and c. Optionally, a diluent.

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